Multipotent adult stem cells, sources thereof, methods of obtaining and maintaining same, methods of differentiation thereof, methods of use thereof and cells derived thereof

ABSTRACT

The present invention relates generally to mammalian multipotent adult stem cells (MASC), and more specifically to methods for obtaining, maintaining and differentiating MASC to cells of multiple tissue types. Uses of MASC in the therapeutic treatment of disease are also provided.

RELATED CASES

This application claims the benefit of U.S. Provisional Application No.60/343,386, filed Oct. 25, 2001, U.S. Provisional Application No.60/310,625, filed Aug. 7, 2001, U.S. Provisional Application No.60/269,062, filed Feb. 15, 2001, U.S. Provisional Application No.60/268,786, filed Feb. 14, 2001, which are hereby incorporated byreference for all purposes. Applicants also claim priority of WO01/11011, 60/147,324 and 60/164,650 and these applications are herebyincorporated by reference into this text; any teachings therein may beused in the practice of this invention. The present application is acontinuation-in-part of WO 01/11011, which is attached herein atAppendix 1 and is part of the present application. Documentsincorporated by reference into this text are not admitted to be priorart.

FIELD OF THE INVENTION

The present invention relates generally to mammalian multipotent adultstem cells (MASC), and more specifically to methods for obtaining,maintaining and differentiating MASC. Uses of MASC in the therapeutictreatment of disease are also provided.

BACKGROUND OF THE INVENTION

Organ and tissue generation from stem cells, and their subsequenttransplantation provide promising treatments for a number ofpathologies, making stem cells a central focus of research in manyfields. Stem cell technology provides a promising alternative therapyfor diabetes, Parkinson's disease, liver disease, heart disease, andautoimmune disorders, to name a few. However, there are at least twomajor problems associated with organ and tissue transplantation.

First, there is a shortage of donor organs and tissues. As few as 5percent of the organs needed for transplant in the United States aloneever become available to a recipient (Evans, et al. 1992). According tothe American Heart Association, only 2,300 of the 40,000 Americans whoneeded a new heart in 1997 received one. The American Liver Foundationreports that there are fewer than 3,000 donors for the nearly 30,000patients who die each year from liver failure.

The second major problem is the potential incompatibility of thetransplanted tissue with the immune system of the recipient. Because thedonated organ or tissue is recognized by the host immune system asforeign, immunosuppressive medications must be provided to the patientat a significant cost-both financially and physically.

Xenotransplantation, or transplantation of tissue or organs from anotherspecies, could provide an alternative means

to overcome the shortage of human organs and tissues.Xenotransplantation would offer the advantage of advanced planning. Theorgan could be harvested while still healthy and the patient couldundergo any beneficial pretreatment prior to transplant surgery.Unfortunately, xenotransplantation does not overcome the problem oftissue incompatibility, but instead exacerbates it. Furthermore,according to the Centers for Disease Control, there is evidence thatdamaging viruses cross species barriers. Pigs have become likelycandidates as organ and tissue donors, yet cross-species transmission ofmore than one virus from pigs to humans has been documented. Forexample, over a million pigs were recently slaughtered in Malaysia in aneffort to contain an outbreak of Hendra virus, a disease that wastransmitted to more than 70 humans with deadly results (Butler, D.1999).

Stem Cells: Definition and Use

The most promising source of organs and tissues for transplantation,therefore, lies in the development of stem cell technology.Theoretically, stem cells can undergo self-renewing cell division togive rise to phenotypically and genotypically identical daughters for anindefinite time and ultimately can differentiate into at least one finalcell type. By generating tissues or organs from a patient's own stemcells, or by genetically altering heterologous cells so that therecipient immune system does not recognize them as foreign, transplanttissues can be generated to provide the advantages associated withxenotransplantation without the associated risk of infection or tissuerejection.

Stem cells also provide promise for improving the results of genetherapy. A patient's own stem cells could be genetically altered invitro, then reintroduced in vivo to produce a desired gene product.These genetically altered stem cells would have the potential to beinduced to differentiate to form a multitude of cell types forimplantation at specific sites in the body, or for systemic application.Alternately, heterologous stem cells could be genetically altered toexpress the recipient's major histocompatibility complex (MHC) antigen,or no MHC antigen, allowing transplantion of cells from donor torecipient without the associated risk of rejection.

Stem cells are defined as cells that have extensive proliferationpotential, differentiate into several cell lineages, and repopulatetissues upon transplantation. The quintessential stem cell is theembryonic stem (ES) cell, as it has unlimited self-renewal andmultipotent differentiation potential (Thomson, J. et al. 1995; Thomson,J. A. et al. 1998; Shamblott, M. et al. 1998; Williams, R. L. et al.1988; Orkin, S. 1998; Reubinoff, B. E., et al. 2000). These cells arederived from the inner cell mass of the blastocyst (Thomson, J. et al.1995; Thomson, J. A. et al. 1998; Martin, G. R. 1981), or can be derivedfrom the primordial germ cells from a post-implantation embryo(embryonal germ cells or EG cells). ES and EG cells have been derivedfrom mouse, and more recently also from non-human primates and humans.When introduced into mouse blastocysts, ES cells can contribute to alltissues of the mouse (animal) (Orkin, S. 1998). Murine ES cells aretherefore pluripotent. When transplanted in post-natal animals, ES andEG cells generate teratomas, which again demonstrates theirmultipotency. ES (and EG) cells can be identified by positive stainingwith the antibodies to stage-specific embryonic antigens (SSEA) 1 and 4.

At the molecular level, ES and EG cells express a number oftranscription factors highly specific for these undifferentiated cells.These include oct-4 and Rex-1, leukemia inhibitory factor receptor(LIF-R). The transcription factors sox-2 and Rox-1 are expressed in bothES and non-ES cells. Oct-4 is expressed in the pregastrulation embryo,early cleavage stage embryo, cells of the inner cell mass of theblastocyst, and embryonic carcinoma (EC) cells. In the adult animal,oct-4 is only found in germ cells.

Oct-4, in combination with Rox-1, causes transcriptional activation ofthe Zn-finger protein Rex-1, and is also required for maintaining ES inan undifferentiated state. The oct-4 gene is down-regulated when cellsare induced to differentiate in vitro. Several studies have shown thatoct-4 is required for maintaining the undifferentiated phenotype of EScells, and that it plays a major role in determining early steps inembryogenesis and differentiation. Sox-2, is required with oct-4 toretain the undifferentiated state of ES/EC and to maintain murine, butnot human, ES cells. Human or murine primordial germ cells requirepresence of LIF. Another hallmark of ES cells is presence of high levelsof telomerase, which provides these cells with an unlimited self-renewalpotential in vitro.

Stem cells have been identified in most organs or tissues. The bestcharacterized is the hematopoietic stem cell (HSC). Thismesoderm-derived cell has been purified based on cell surface markersand functional characteristics. The HSC, isolated from bone marrow (BM),blood, cord blood, fetal liver and yolk sac, is the progenitor cell thatgenerates blood cells or following translation reinitiates multiplehematopoietic lineages and can reinitiate hematopoiesis for the life ofa recipient. (See Fei, R., et al., U.S. Pat. No. 5,635,387; McGlave, etal., U.S. Pat. No. 5,460,964; Simmons, P., et al., U.S. Pat. No.5,677,136; Tsukamoto, et al., U.S. Pat. No. 5,750,397; Schwartz, et al.,U.S. Pat. No. 759,793; DiGuisto, et al., U.S. Pat. No. 5,681,599;Tsukamoto, et al., U.S. Pat. No. 5,716,827; Hill, B., et al. 1996.) Whentransplanted into lethally irradiated animals or humans, HSCs canrepopulate the erythroid, neutrophil-macrophage, megakaryocyte andlymphoid hemopoietic cell pool. In vitro, hemopoietic stem cells can beinduced to undergo at least some self-renewing cell divisions and can beinduced to differentiate to the same lineages as is seen in vivo.Therefore, this cell fulfills the criteria of a stem cell. Stem cellswhich differentiate only to form cells of hematopoietic lineage,however, are unable to provide a source of cells for repair of otherdamaged tissues, for example, heart or lung tissue damaged by high-dosechemotherapeutic agents.

A second stem cell that has been studied extensively is the neural stemcell (NSC) (Gage F. H. 2000; Svendsen C. N. et al, 1999; Okabe S. et al.1996). NSCs were initially identified in the subventricular zone and theolfactory bulb of fetal brain. Until recently, it was believed that theadult brain no longer contained cells with stem cell potential. However,several studies in rodents, and more recently also non-human primatesand humans, have shown that stem cells continue to be present in adultbrain. These stem cells can proliferate in vivo and continuouslyregenerate at least some neuronal cells in vivo. When cultured ex vivo,NSCs can be induced to proliferate, as well as to differentiate intodifferent types of neurons and glial cells. When transplanted into thebrain, NSCs can engraft and generate neural cells and glial cells.Therefore, this cell too fulfills the definition of a stem cell, albeita hematopoetic stem cell.

Clarke et al. reported that NSCs from Lac-Z transgenic mice injectedinto murine blastocysts or in chick embryos contribute to a number oftissues of the chimeric mouse or chicken embryo (Clarke, D. L. et al.2000). LacZ-expressing cells were found with varying degree ofmosaicism, not only in the central nervous system, but also inmesodermal derivatives as well as in epithelial cells of the liver andintestine but not in other tissues, including the hematopoietic system.These studies therefore suggested that adult NSCs may have significantlygreater differentiation potential than previously realized but still donot have the pluripotent capability of ES or of the adult derivedmultipotent adult stem cells (MASC) described in Furcht et al.(International Application No. PCT/US00/21387) and herein. The termsMASC, MAPC and MPC can also be used interchagably to describe adultderived multipotent adult stem cells.

Therapies for degenerative and traumatic brain disorders would besignificantly furthered with cellular replacement therapies. NSC havebeen identified in the sub-ventricular zone (SVZ) and the hippocampus ofthe adult mammalian brain (Ciccolini et al., 1998; Morrison et al.,1999; Palmer et al., 1997; Reynolds and Weiss, 1992; Vescovi et al.,1999) and may also be present in the ependyma and other presumednon-neurogenic areas of the brain (Doetsch et al., 1999; Johansson etal., 1999; Palmer et al., 1999). Fetal or adult brain-derived NSC can beexpanded ex vivo and induced to differentiate into astrocytes,oligodendrocytes and functional neurons (Ciccolini et al., 1998;Johansson et al., 1999; Palmer et al., 1999; -Reynolds et al., 1996;Ryder et al., 1990; Studer et al., 1996; Vescovi et al., 1993). In vivo,undifferentiated NSC cultured for variable amounts of time differentiateinto glial cells, GABAergic and dopaminergic neurons (Flax et al., 1998;Gage et al., 1995; Suhonen et al., 1996). The most commonly used sourceof NSC is allogeneic fetal brain, which poses both immunological andethical problems. Alternatively, NSC could be harvested from theautologous brain. As it is not known whether pre-existing neuralpathology will affect the ability of NSC to be cultured and induced todifferentiate into neuronal and glial cells ex vivo, and becauseadditional surgery in an already diseased brain may aggravate theunderlying disease, this approach is less attractive.

The ideal source of neurons and glia for replacement strategies would becells harvestable from adult, autologous tissue different than the brainthat was readily accessible and that can be expanded in vitro anddifferentiated ex vivo or in vivo to the cell type that is deficient inthe patient. Recent reports have suggested that BM derived cells acquirephenotypic characteristics of neuroectodermal cells when cultured invitro under NSC conditions, or when they enter the central nervoussystem (Sanchez-Ramos et al., 2000; Woodbury et al., 2000). Thephenotype of the BM cells with this capability is not known. Thecapacity for differentiation of cells that acquire neuroectodermalfeatures to other cell types is also unknown.

A third tissue specific cell with stem cell properties is themesenchymal stem cell (MSC), initially described by Fridenshtein (1982).MSC, originally derived from the embryonal mesoderm and isolated fromadult BM, can differentiate to form muscle, bone, cartilage, fat, marrowstroma, and tendon. During embryogenesis, the mesoderm develops intolimb-bud mesoderm, tissue that generates bone, cartilage, fat, skeletalmuscle and possibly endothelium. Mesoderm also differentiates tovisceral mesoderm, which can give rise to cardiac muscle, smooth muscle,or blood islands consisting of endothelium and hematopoietic progenitorcells. Primitive mesodermal or MSCs, therefore, could provide a sourcefor a number of cell and tissue types. A number of MSCs have beenisolated. (See, for example, Caplan, A., et al., U.S. Pat. No.5,486,359; Young, H., et al., U.S. Pat. No. 5,827,735; Caplan, A., etal., U.S. Pat. No. 5,811,094; Bruder, S., et al., U.S. Pat. No.5,736,396; Caplan, A., et al., U.S. Pat. No. 5,837,539; Masinovsky, B.,U.S. Pat. No. 5,837,670; Pittenger, M., U.S. Pat. No. 5,827,740;Jaiswal, N., et al., 1997; Cassiede P., et al., 1996; Johnstone, B., etal., 1998; Yoo, et al., 1998; Gronthos, S., 1994).

Of the many MSC that have been described, all have demonstrated limiteddifferentiation to form cells generally considered to be of mesenchymalorigin. To date, the most multipotent MSC reported is the cell isolatedby Pittenger, et al., which expresses the SH2⁺ SH4⁺ CD29⁺ CD44⁺ CD71⁺CD90⁺ CD106⁺ CDI20a⁺ CD124⁻ CD 14⁻ CD34⁻ CD45⁻ phenotype. This cell iscapable of differentiating to form a number of cell types of mesenchymalorigin, but is apparently limited in differentiation potential to cellsof the mesenchymal lineage, as the team who isolated it noted thathematopoietic cells were never identified in the expanded cultures(Pittenger, et al., 1999).

Other tissue-specific stem cells have been identified, includinggastrointestinal stem cells (Potten, C. 1998), epidermal stem cells(Watt, F. 1997), and hepatic stem cells, also termed oval cells (Alison,M. et al., 1998). Most of these are less well characterized.

Compared with ES cells, tissue specific stem cells have lessself-renewal ability and, although they differentiate into multiplelineages, they are not pluripotent. No studies have addressed whethertissue specific cells express the markers described above as seen in EScells. In addition, the degree of telomerase activity in tissue specificor lineage comitted stem cells has not been fully explored, in partbecause large numbers of highly enriched populations of these cells aredifficult to obtain.

Until recently, it was thought that tissue specific stem cells couldonly differentiate into cells of the same tissue. A number of recentpublications have suggested that adult organ specific stem cells may becapable of differentiation into cells of different tissues. However, thetrue nature of these types of cells has not been fully discerned. Anumber of studies have shown that cells transplanted at the time of a BMtransplant can differentiate into skeletal muscle (Ferrari 1998; Gussoni1999). This could be considered within the realm of possibledifferentiation potential of mesenchymal cells that are present inmarrow. Jackson published that muscle satellite cells can differentiateinto hemopoietic cells, again a switch in phenotype within thesplanchnic mesoderm of the embryo (Jackson 1999). Other studies haveshown that stem cells from one embryonic layer (for instance splanchnicmesoderm) can differentiate into tissues thought to be derived duringembryogenesis from a different embryonic layer. For instance,endothelial cells or their precursors detected in humans or animals thatunderwent marrow transplantation are at least in part derived from themarrow donor (Takahashi, 1999; Lin, 2000). Thus, visceral mesoderm andnot splanchnic mesoderm, capabilities such as MSC, derived progeny aretransferred with the infused marrow. Even more surprising are thereports demonstrating both in rodents and humans that hepatic epithelialcells and biliary duct epithelial cells can be seen in recipients thatare derived from the donor marrow (Petersen, 1999; Theise, 2000; Theise,2000). Likewise, three groups have shown that NSCs can differentiateinto hemopoietic cells. Finally, Clarke et al., reported that cells betermed NSCs when injected into blastocysts can contribute to all tissuesof the chimeric mouse (Clarke et al., 2000).

It is necessary to point out that most of these studies have notconclusively demonstrated that a single cell can differentiate intotissues of different organs. Also, stem cells isolated from a givenorgan may not necessarily be a lineage committed cell. Indeed mostinvestigators did not identify the phenotype of the initiating cell. Anexception is the study by Weissman and Grompe, who showed that cellsthat repopulated the liver were present in Lin⁻Thy₁LowSca₁ ⁺ marrowcells, which are highly enriched in HSCs. Likewise, the Mulligan groupshowed that marrow Sp cells, highly enriched for HSC, can differentiateinto muscle and endothelium, and Jackson et al. showed that muscle Spcells are responsible for hemopoietic reconstitution (Gussoni et al.,1999).

Transplantation of tissues and organs generated from heterologous EScells requires either that the cells be further genetically modified toinhibit expression of certain cell surface markers, or that the use ofchemotherapeutic immune suppressors continue in order to protect againsttransplant rejection. Thus, although ES cell research provides apromising alternative solution to the problem of a limited supply oforgans for transplantation, the problems and risks associated with theneed for immunosuppression to sustain transplantation of heterologouscells or tissue would remain. An estimated 20 immunologically differentlines of ES cells would need to be established in order to provideimmunocompatible cells for therapies directed to the majority of thepopulation.

Using cells from the developed individual, rather than an embryo, as asource of autologous or from tissue typing matched allogeneic stem cellswould mitigate or overcome the problem of tissue incompatibilityassociated with the use of transplanted ES cells, as well as solve theethical dilemma associated with ES cell research. The greatestdisadvantage associated with the use of autologous stem cells for tissuetransplant thus far lies in their relatively limited differentiationpotential. A number of stem cells have been isolated fromfully-developed organisms, particularly humans, but these cells,although reported to be multipotent, have demonstrated limited potentialto differentiate to multiple cell types.

Thus, even though stem cells with multiple differentiation potentialhave been isolated previously by others and by the present inventors, aprogenitor cell with the potential to differentiate into a wide varietyof cell types of different lineages, including fibroblasts, hepatic,osteoblasts, chondrocytes, adipocytes, skeletal muscle, endothelium,stroma, smooth muscle, cardiac muscle and hemopoietic cells, has notbeen described. If cell and tissue transplant and gene therapy are toprovide the therapeutic advances expected, a stem cell or progenitorcell with the greatest or most extensive differentiation potential isneeded. What is needed is the adult equivalent of an ES cell.

BM, muscle and brain are the three tissues in which cells with apparentgreater plasticity than previously thought have been identified. BMcontains cells that can contribute to a number of mesodermal (Ferrari G.et al., 1998; Gussoni E. et al., 1999; Rafii S. et al., 1994; Asahara T.et al., 1997; Lin Y. et al., 2000; Orlic D. et al., 2001; Jackson K. etal., 2001) endodermal (Petersen B. E. et al., 1999; Theise, N. D. etal., 2000; Lagasse E. et al., 2000; Krause D. et al., 2001) andneuroectodermal (Mezey D. S. et al., 2000; Brazelton T. R., et al.,2000, Sanchez-Ramos J. et al., 2000; Kopen G. et al., 1999) and skin(Krause, D. et al., 2001) structures. Cells from muscle may contributeto the hematopoietic system (Jackson K. et al., 1999; Seale P. et al.,2000). There is also evidence that NSC may differentiate intohematopoietic cells (Bjornson C. et al., 1999; Shih C. et al., 2001),smooth muscle myoblasts (Tsai R. Y. et al., 2000) and that NSC give riseto several cell types when injected in a mouse blastocyst (Clarke, D. L.et al., 2000).

The present study demonstrates that cells with multipotent adultprogenitor characteristics can be culture-isolated from multipledifferent organs, namely BM, muscle and the brain. The cells have thesame morphology, phenotype, in vitro differentiation ability and have ahighly similar expressed gene profile.

SUMMARY OF THE INVENTION

The present invention is a multipotent adult stem cell (MASC) isolatedfrom a mammal, preferably mouse, rat or human. The cell is derived froma non-embryonic organ or tissue and has the capacity to be induced todifferentiate to form at least one differentiated cell type ofmesodermal, ectodermal and endodermal origin. In a preferred embodiment,the organ or tissue from which the MASC are isolated is bone marrow,muscle, brain, umbilical cord blood or placenta.

Examples of differentiated cells that can be derived from MASC areosteoblasts, chondrocytes, adipocytes, fibroblasts, marrow stroma,skeletal muscle, smooth muscle, cardiac muscle, occular, endothelial,epithelial, hepatic, pancreatic, hematopoietic, glial, neuronal oroligodendrocytes. Differentiation can be induced in vivo or ex vivo.

The MASC of the present invention is also summarized as a cell thatconstitutively expresses oct4 and high levels of telomerase and isnegative for CD44, MHC class I and MHC class II expression. As a methodof treatment, this cell administered to a patient in a therapeuticallyeffective amount. A surprising benefit of this treatment is that noteratomas are formed in vivo.

An object of the invention is to produce a normal, non-human animalcomprising MASC. Preferably, the animal is chimeric.

Another embodiment of the invention is a composition comprising apopulation of MASC and a culture medium that expands the MASCpopulation. It is advantageous in some cases for the medium to containepidermal growth factor (EGF), platelet derived growth factor (PDGF) andleukemia inhibitory factor (LIF).

The present invention also provides a composition comprising apopulation of fully or partially purified MASC progeny. The progeny canhave the capacity to be further differentiated, or can be terminallydifferentiated.

In a preferable embodiment, the progeny are of the osteoblast,chondrocyte, adipocyte, fibroblast, marrow stroma, skeletal muscle,smooth muscle, cardiac muscle, occular, endothelial, epithelial,hepatic, pancreatic, hematopoietic, glial, neuronal or oligodendrocytecell type.

The present invention also provides a method for isolating andpropagating MASC by obtaining tissue from a mammal, establishing apopulation of adherent cells, depleting the population of CD45⁺ cells,recovering CD45⁻ cells and culturing them under expansion conditions toproduce an expanded cell population. An object of the present invention,therefore, is to produce an expanded cell population obtained by thismethod.

An aspect of the invention is a method for differentiating MASC ex vivoby isolating and propagating them, and then culturing the propagatedcells in the presence of desired differentiation factors. The preferreddifferentiation factors are basic fibroblast growth factor (bFGF),vascular endothelial growth factor (VEGF), dimethylsulfoxide (DMSO) andisoproterenol; or fibroblast growth factor4 (FGF4) and hepatocyte growthfactor (HGF). Another aspect of the invention is the differentiated cellitself.

The invention includes a method for differentiating MASC in vivo, byisolating and expanding them, and then administering the expanded cellpopulation to a mammalian host, wherein said cell population isengrafted and differentiated in vivo in tissue specific cells, such thatthe function of a cell or organ, defective due to injury, geneticdisease, acquired disease or iatrogenic treatments, is augmented,reconstituted or provided for the first time. Using this method, theMASC can undergo self-renewal in vivo.

A further aspect of the invention is a differentiated cell obtained byex vivo or in vivo differentiation. In a preferred embodiment, thedifferentiated cell is ectoderm, mesoderm or endoderm. In anotherpreferred embodiment, the differentiated cell is of the osteoblast,chondrocyte, adipocyte, fibroblast, marrow stroma, skeletal muscle,smooth muscle, cardiac muscle, occular, endothelial, epithelial,hepatic, pancreatic, hematopoietic, glial, neuronal or oligodendrocytecell type.

An important application of this technology is the method of treating apatient by administering a therapeutically effective amount of MASC ortheir progeny. The progeny can either have the capacity to be furtherdifferentiated, or can be terminally differentiated. An unexpectedbenefit of this approach is that the need for pretreatment and/or posttreatment of the patient with irradiation, chemotherapy,immunosuppressive agents or other drugs or treatments is reduced oreliminated. The induction of tolerance before or during treatment isalso not required.

Such treatment can treat a variety of diseases and conditions, includingcancer, cardiovascular disease, metabolic disease, liver disease,diabetes, hepatitis, hemophilia, degenerative or traumatic neurologicalconditions, autoimmune disease, genetic deficiency, connective tissuedisorders, anemia, infectious disease and transplant rejection.

MASC or their progeny are administered via localized injection,including catheter administration, systemic injection, parenteraladministration, oral administration, or intrauterine injection into anembryo. Administration can be in conjunction with a pharmaceuticallyacceptable matrix, which may be biodegradable.

MASC or their progeny, administered to a patient, alter the immunesystem to resist viral, bacterial or fungal infection.

Surprisingly, teratomas are not formed when MASC or their progeny areadminstered to a patient.

When administered to a patient, MASC or their progeny also are able toaugment, reconstitute or provide for the first time the function of acell or organ defective due to injury, genetic disease, acquired diseaseor iatrogenic treatments. The organ is any of bone marrow, blood,spleen, liver, lung, intestinal tract, brain, immune system, circulatorysystem, bone, connective tissue, muscle, heart, blood vessels, pancreas,central nervous system, peripheral nervous system, kidney, bladder,skin, epithelial appendages, breast-mammary glands, fat tissue, andmucosal surfaces including oral esophageal, vaginal and anal. Examplesof diseases treatable by this method are cancer, cardiovascular disease,metabolic disease, liver disease, diabetes, hepatitis, hemophilia,degenerative or traumatic neurological conditions, autoimmune disease,genetic deficiency, connective tissue disorders, anemia, infectiousdisease and transplant rejection.

The MASC or their progeny home to one or more organs in the patient andare engrafted therein such that the function of a cell or organ,defective due to injury, genetic disease, acquired disease or iatrogenictreatments, is augmented, reconstituted or provided for the first time,which is surprising and unexpected. In a preferred embodiment, theinjury is ischemia or inflammation.

In another preferred embodiment, the MASC or their progeny enhanceangiogenesis.

In an additional aspect of the invention, MASC or their progeny aregenetically transformed to deliver a therapeutic agent, preferably anantiangiogenic agent.

The invention provides a therapeutic composition comprising MASC and apharmaceutically acceptable carrier, wherein the MASC are present in anamount effective to produce tissue selected from the group consisting ofbone marrow, blood, spleen, liver, lung, intestinal tract, brain, immunesystem, bone, connective tissue, muscle, heart, blood vessels, pancreas,central nervous system, kidney, bladder, skin, epithelial appendages,breast-mammary glands, fat tissue, and mucosal surfaces including oralesophageal, vaginal and anal.

The invention further provides a therapeutic method for restoring organ,tissue or cellular function to a patient comprising the steps ofremoving MASC from a mammalian donor, expanding MASC to form an expandedpopulation of undifferentiatied cells, and adminstering the expandedcells to the patient, wherein organ, tissue or cellular function isrestored. The restored function may be enzymatic or genetic. In apreferred embodiment, the mammalian donor is the patient.

The invention provides a method of inhibiting the rejection of aheterologous MASC transplanted into a patient comprising the steps ofintroducing into the MASC, ex vivo, a nucleic acid sequence encoding therecipient's MHC antigen operably linked to a promotor, wherein the MHCantigen is expressed by the MASC and transplanting the MASC into thepatient, wherein MHC antigen is expressed at a level sufficient toinhibit the rejection of the transplanted MASC. The patient is of thesame species or another mammalian species as the donor of the MASC.

An alternative method of inhibiting the rejection of a heterologous MASCtransplanted into a patient comprises transgenically knocking outexpression of MHC antigen in the MASC and transplanting the transgenicMASC into the patient MHC antigen is not expressed by the MASC andrejection of the transplanted cells is inhibited.

An object of the invention is a method of generating blood or individualblood components ex vivo by the process of isolating MASC anddifferentiating the MASC to form blood or blood components. Preferably,the individual blood components are red blood cells, white blood cellsor platelets.

Another aspect of the invention is a method of drug discovery comprisingthe steps of analyzing the genomic or proteomic makeup of MASC or theirprogeny, employing analysis thereof via bioinformatics and/or computeranalysis using algorithms, and assembling and comparing new data withknown databases to compare and contrast these.

A further aspect is a method of identifying the components of adifferentiation pathway comprising the steps of analyzing the genomic orproteomic makeup of MASC, inducing differentiation of MASC in vitro orin vivo, analyzing the genomic or proteomic makeup of intermediary cellsin the differentiation pathway, analyzing the genomic or proteomicmakeup of terminally differentiated cells in the differentiationpathway, using bioinformatics and/or algorithms to characterize thegenomic or proteomic makeup of MASC and their progeny, and comparing thedata obtained in (e) to identify the components of the pathway. Usingthis method, differentiation that occurs in vitro can be compared withdifferentiation that occurs in vivo such that fundamental differencesbetween the two systems can be characterized.

The invention provides a method of generating products in vitro thathave therapeutic, diagnostic or research utility by identifying theproducts in MASC and isolating the products from MASC. In a preferredembodiment, the products are proteins, lipids, complex carbohydrates,DNA or RNA.

Included in the invention is a method of inducing, in a mammal,tolerance to an antigen administered to said mammal, the methodcomprising the step of administering to said mammal, after orsimultaneously with the administration of said antigen, an effectiveamount of MASC or their progeny so that said mammal's humoral immuneresponse to a subsequent challenge with said antigen is suppressed.

Also included is a method for removing toxins from the blood of apatient comprising contacting blood ex vivo with MASC derived cells,wherein said cells line a hollow, fiber based device. In a preferredembodiment, the cells are kidney or liver cells.

An object of the invention is a method for delivering therapeuticproducts to a patient comprising contacting the blood of said patient exvivo with MASC or their progeny, wherein said MASC or their progeny aregenetically transformed to deliver a therapeutic agent.

A further object is a method for testing the toxicity of a drugcomprising contacting MASC or their progeny ex vivo with said drug andmonitoring cell survival. In a preferred embodiment, the progeny areselected from the group consisting of hepatic, endothelial, epithelialand kidney.

BRIEF DESCRIPTION OF DRAWINGS

The following Detailed Description, given by way of example, but notintended to limit the invention to specific embodiments described, maybe understood in conjunction with the accompanying drawings,incorporated herein by reference, in which:

FIG. 1 shows a graphical illustration of the expansion potential of bonemarrow (BM), muscle and brain derived MASC.

FIG. 2 shows a scatter plot representing gene expression in (A) muscleand brain MASC and (B) bone marrow and muscle MASC.

FIG. 3 shows a graphical illustration of FACS analysis ofundifferentiated MASC and MASC cultured with VEGF. The plots showisotype control IgG staining profile (thin line) vs. specific antibodystaining profile (thick line). Panel A shows the phenotype ofundifferentiated MASC. MASC express low levels of β2-microglobulin,Flk1, Flt 1 and AC 133, but do not stain with any of the otheranti-endothelial markers; panel B shows the phenotype of MASC culturedfor 14 days with 10 ng/mL VEGF. MASC express low levels most markersassociated with endothelial cells, but lost expression of AC 133; andpanel C shows phenotype of MASC cultured or 3-9 days with 10 ng/mL VEGF.MASC lose expression of AC 133 by day 3 of culture with VEGF, acquireexpression of Tek and VE-cadherin by day 3, Tie, vWF, CD34 and HIP12 byday 9.

FIG. 4 shows a photomicrograph of engraftment and in vivodifferentiation of mMASC. Slides were examined by fluorescence orconfocal microscopy. Panels A, G, J, N, Q and S represent identicallystained tissues of control NOD-SCID animals that were not injected withmMASC. Panels A-F show a photomicrograph of bone marrow (BM) cytospinfrom a control (A) and study (B-F) animal stained with antiβ-gal-FITCantibody and PE-conjugated antibodies to various hematopoietic antigens.A-B: CD45, C: CD19, D: MAC1, E: GR1, F: TER119 and DAPI; panels G-Ishows a photomicrograph of a spleen section from a control (G) and studyanimal (H, I) stained with anti-β-gal-FITC antibody and anti-CD45-PEantibody. Donor derived anti-β-gal⁺ cells are seen in clusters. H is 10×and I are 60× magnifications; panels J-M shows a photomicrograph of aliver, section from a control mouse (J) and study animal (K-M) stainedwith anti-β-gal-FITC. J-L are co-stained withmouse-anti-CK-18/anti-mouse-Cy5 plus CD45-PE and M with mouseanti-albumin/anti-mouse Cy3 antibodies. J-K, L and M are 20×, 60× and10× magnifications respectively; panels N-P show a photomicrograph of anintestine section from a control mouse and study animal (O-P), stainedwith anti-β-gal-FITC plus mouse-anti-pan-CK/anti-mouse-Cy5 antibodies(N-P). N and P are costained with CD45-PE antibodies. β-gal⁺Pan-CK⁺CD45⁻ epithelial cells covered 50% (solid arrow, panel P) of thecircumference of villi. Pan-CK⁻/β-gal⁺ cells in the core of the villi(open arrow-panel O) co-stained for CD45 (P); panels Q-R show aphotomicrograph of a lung section from a control mouse (O) and studyanimal (R) stained with anti-β-gal-FITC plusmouse-anti-pan-CK/anti-mouse-Cy5 plus CD45-PE antibodies. Several β-gal⁺pan-CK⁺ donor cells are seen lining the alveoli of the recipient animal(R). CD45⁺/pan-CK⁻ cells of hematopoietic origin are seen distinctlyfrom the epithelial cells; and panels S-T show a photomicrograph of ablood vessel section from a control mouse (S) and thymic lymphoma thatdeveloped in a study animal 16 weeks after transplantation (T) stainedwith anti-β-gal-FITC, anti-vWF-PE and TO-PRO3. β-gal⁺ donor cellsdifferentiated into vWF⁺ endothelial cells in the thymic lymphoma whichis of recipient origin, as the tumor cells did not stain with anti-β-Galantibodies.

FIG. 5 shows immunohistochemical evaluation of MASC-derived endothelialcells using confocal fluorescence microscopy. (a) MASC grown for 14 daysin VEGF. Typical membrane staining is seen for the adhesion receptor,αvβ5, and for the adherens junction proteins, ZO-1, β- and γ-catenin.Scale bar=50 μm. (b) Morphology in bright field of MASC at day 0 (upperpanel) and day 21 (lower panel) after VEGF treatment. Bar=25 μm.

FIG. 6 shows a photomicrograph of MASC derived endothelial cells. PanelA shows histamine-mediated release of vWF from MASC-derived endothelium.Staining with antibodies against myosin shows cytoskeletal changes withincreased numbers of myosin stress fibers, and widening of gap junctions(Arrows) (Representative example of 3 experiments). Scale bar=60 μm;panel B shows MASC-derived endothelium takes up a-LDL. After 7 days,cells expressed Tie-1, but again did not take up a-LDL. However,acquisition of expression of vVWF on day 9 was associated with uptake ofaLDL (representative example of 10 experiments). Scale bar=100 μm; andpanel C shows vascular tube formation by MASC-derived endothelium. After6 h, typical vascular tubes could be seen. (Representative example of 6experiments). Scale bar=200 μm

FIG. 7 shows a graphical illustration of FACS analysis of MASC derivedendothelial cells. The Plots show isotype control IgG staining profile(thin line) vs. specific antibody staining profile (thick line)(Representative example of >3 experiments). Number above plots is theMean Fluorescence Intensity (MFI) for the control IgG staining and thespecific antibody staining. Panel A shows hypoxia upregulates Flk1 andTek expression on MASC-derived endothelial cells analyzed by flowcytometry; panel B shows that hypoxia upregulates VEGF production byMASC-derived endothelial cells. VEGF levels were measured by ELISA andthe results are shown as Mean±SEM of 6 experiments; and panel C showsthat IL-1a induces expression of class II HLA antigens and increasesexpression of adhesion receptors. Plots show isotype control IgGstaining profile (thin line) vs. specific antibody staining profile(thick line) (Representative example of 3 experiments). Number aboveplots shows MFI for the control IgG staining and the specific antibodystaining.

FIG. 8 shows a photomicrograph of human MASC derived endothelial cells.Panels C-F show the 3-D reconstructed figures for either anti-humanβ2-microglobulin-FITC (panel C) or anti-mouse-CD31-FITC (panel D) andmerging of the two (Panel E), anti-vWF-Cy3 (panel F), and merging of thethree staining patterns (Panel G). Panels A and B show the confocalimage of a single slice stained with either anti-humanβ2-microglobulin-FITC and anti-vWF-Cy3, or anti-mouse-CD31-Cy5 andanti-vWF-Cy3. Scale bar=100 μm. Panel H shows wound healing resulting ina highly vascularized area in the punched ear stained withanti-β2-microglobulin-FITC and anti-vWF in mice injected with humanMASC-derived endothelial cells (Top panel) or human foreskin fibroblasts(Bottom panel). Scale bar=20 μm. C=Cartilage. D=dermis. Panel I showsthat tumor angiogenesis is derived from endothelial cells generated invivo from MASC resulting in a highly vascularized area in the tumorstained with anti-X32-microglobulin-FITC, anti-vWF and TOPRO-3. Scalebar=20 μm.

FIG. 9 shows spiking behavior and expressed voltage-gated sodiumcurrents in hMSC derived neuron-like cells. Panel A shows aphotomicrograph of cultured hMSC-derived neurons that showed spikingbehavior and expressed voltage-gated sodium currents (the shadow of thepipette points to the cell). Panel B shows graphical illustrations ofcurrent-clamp recordings from a hMSC derived neuron. Panel C showsgraphical illustrations of leak-subtracted current traces from the samehMSC derived neuron.

FIG. 10 shows quantitative RT-PCR and Western blot analysis confirmingthe hepatocyte-like phenotype. Panels A and B show mMASC (A) and hMASC(B) cultured on Matrigel™ with FGF4 and HGF or FGF4 alone for 21 and 28days respectively. For αFP, Cyp2b9 and Cyp2b13, numbers under the blotsare relative to mRNA from liver, as no transcripts were detected inundifferentiated MASC. Li=mouse or human liver mRNA; NT=no-template.Representative example of 5 mouse and 1 human studies, Panel C showshMASC (B) cultured on Matrigel™ with FGF4 and HGF or FGF4 alone for 21days. FH=FGF4 and HGF-induced hMASC on Matrigel™, Huh=Huh7 cell lineused as control.

FIG. 11 shows a photomicrograph of hepatocyte-like cells. MASC inducedby FGF4 produce glycogen. Glycogen storage is seen as accumulation ofdark staining (Representative example of 3 studies). Scale bar=25 μm.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, the following terms shall have the following meanings:

“Expansion” shall mean the propogation of a cell withoutdifferentiation.

“Intermediary cells” are cells produced during differentiation of a MASCthat have some, but not all, of the characteristics of MASC or theirterminally differentiated progeny. Intermediary cells may be progenitorcells which are committed to a specific pathway, but not to a specificcell type.

“Normal” shall mean an animal that is not diseased, mutated ormalformed, i.e., healthy animals.

“Self-renewal” shall mean the ability of cells to propagate without theaddition of external stimulation. The presence of cytokines or othergrowth factors produced locally in the tissue or organ shall notconstitute external stimulation.

“Home” shall mean the ability of certain MASC or their progeny tomigrate specifically to sites where additional cells may be needed.

“Knocking out expression” shall mean the elimination of the function ofa particular gene.

As used herein, “genomic or proteomic makeup” shall mean the gene orprotein components of a given cell.

“High levels of telomerase activity” can be correlated to the two-foldlevel observed in the immortal human cell line MCF7. Soule et al. (1973)J. Cancer Inst. 51:1409-1416.

Application of this Technology

MASC technology could be used to replace damaged, diseased,dysfunctional or dead cells in the body of a mammal. Furthermore thesecells could be injected into the host using autologous or allogeneiccells with or without nature or artificial supports, matrices orpolymers to correct for loss of cells, abnormal function or cells ororgans e.g. genetic such as mutations of genes affecting a proteinfunction such as sickle cell disease, hemophilia or “storage diseases”where products accumulate in the body because of faulty processing, e.g.Guacher's, Neiman Pick's, mucopolysaccharidosis etc. Examples ofrestitution of dying or dead cells would be the use of MASC or theirdifferentiated progeny in the treatment of macular degeneration andother neurodegenerative diseases.

Given the ability to have these MASC to “home” to and incorporate intoorgans/tissues of a host animal proliferate and differentiation theycould potentially be used to provide new endothelial cells to anischemic heart and also myocardial cells themselves, numerous otherexamples exist.

There may be medical circumstances where transient benefits to a tissueor organs function could have desirable effects. For example, there arenow cases with liver failure patients hooked up to a bioartificialliver, which was sufficient to allow for the recovery of normal liverfunction, obviating the need for a liver transplant. This is a seriousunmet medical need, for example in one liver disease alone—hepatitis C.There are 4-5 million Americans currently infected with hepatitis C andthere are estimates that 50% of these people will get cirrhosis and needa liver transplant. This is a huge public health problem that is beggingfor a remedy. Hepatocytes, derived from autologous or allogeneic MASC,can be transplanted in this or other liver diseases. Such transplantsmay either transiently provide liver function to allow recovery of therecipient's own liver cells or permanently repopulate a damaged liver toallow recovery of normal liver function via the donor cells.

In addition to many cell therapies where the undifferentiated MASC areadministered to a human or other mammal to then differentiate intospecific cells in the donor, the progeny of the MASC could bedifferentiated ex vivo and then be administered as purified or evenmixtures of cells to provide a therapeutic benefit. These MASC in theundifferentiated state could also be used as carriers or vehicles todeliver drugs or molecules of therapeutic benefit. This could be totreat any one of a number of diseases including but not limited tocancer, cardiovascular, inflammatory, immunologic, infections, etc. Soby example, a cell perhaps an endothelial cell expressing a novel orhigh levels of an angiogenic molecule could be administered to a patientwhich would be incorporated into existing blood vessels to promoteangiogenesis, for example in the heart; correspondingly one could haveendothelial cells producing molecules that might suppress angiogenesisthat would be incorporated into blood cells and inhibit their furtherformation for example in diabetic retinopathy or in cancer where newblood vessel formation is key to the pathogenesis, spread and extent ofthe disease.

The ability to populate the BM and to form blood ex vivo has an untolduse for important medical applications. For example regarding ex vivoproduction of blood, the transfusion of blood and blood products aroundthe world is still performed with variable safety because oftransmission of infectious agents. Blood transfusions have lead to HIV,hepatitis C and B, and now the impending threat of Mad Cow or CJD,Creuzfeldt-Jakob disease. The ability to produce blood in vitro,especially red blood cells, could provide a safe and reliablealternative to collection of blood from people. It might never fullyreplace blood collection from donors. hMASC or their hematopoieticprogeny could be placed in animals in utero such as sheep which couldform human hematopoietic cells and serve as a source for human bloodcomponents or proteins of therapeutic utility. The same could be truefor hepatocytes, islets or many other cell types but would provide analternative to producing human cells in vitro and use the animals asfactories for the cells. It could also assist in blood shortages thatare predicted to occur. hMASC could also conceivably be transplantedinto a human embryo to correct any one of a number of defects.

Because these MASC can give rise to clonal populations of specificallydifferentiated cells they are a rich platform for drug discovery. Thiswould involve doing gene expression, analyzing gene expression,discovery of new genes activated patterns of activation, proteomics andpatterns of protein expression and modification surrounding this. Thiswould be analyzed with bioinformatics, using data bases and algorithmsfor analyzing these data compared to publicly available or proprietarydata bases. The information of how known drugs or agents might act couldbe compared to information derived from MASC, their differentiatedprogeny and from a population of people which could be available.Pathways, targets, and receptors could be identified. New drugs,antibodies or other compounds could be found to produce a biologicallydesirable responses. Correspondingly, the MASC and their differentiatedprogeny could be used as monitors for undesirable responses, coupledwith databases, bioinformatics and algorithms.

These MASC derived from human, mouse, rat or other mammals appear to bethe only normal, non-malignant, somatic cell (non germ cell) known todate to express very high levels of telomerase even in late passagecells. The telomeres are extended in MASC and they are karyotypicallynormal. Because MASC injected into a mammal, home to multiple organs,there is the likelihood that newly arrived MASC in a particular organcould be self renewing. As such, they have the potential to repopulatean organ not only with themselves but also with self renewingdifferentiated cell types that could have been damaged, died, orotherwise might have an abnormal function because of genetic or acquireddisease.

For example in type I diabetes there is a progressive loss of insulinproducing beta cells in the pancreatic islets. In various renal diseasesthere is progressive loss of function and in some cases obliteration ofglomerulus. If in the case of diabetes, MASC or differentiated progenymight home to the pancreas and themselves or via interaction withendogenous cells within the pancreas, induce islets to be formed. Thiswould have an ameliorating impact on diabetes. Ultimately conditions,agents or drugs might be found to in vivo control, i.e. promote orinhibit their self renewing capability of the MASC and control, orenhance or inhibit the movement to differentiated progeny, e.g., isletprecursors, hepatocyte precursors, blood precursors, neural and/orcardiac precursors using MASC one will likely find pathways, methods ofactivation and control that might induce endogenous precursor cellswithin an organ to proliferate and differentiation.

This same ability to repopulate a cellular tissue or organ compartmentand self renew and also differentiate could have numerous uses and be ofunprecedented usefulness to meet profound unmet medical needs. So forexample certain genetic diseases where there are enzyme deficiencieshave been treated by BM transplantation. Often times this may help butnot cure the complications of the disease where residual effects of thedisease might persist in the brain or bones or elsewhere, MASC andgenetically engineered MASC offer the hope to ameliorate numerousgenetic and acquired diseases. They will also be useful for diagnosticand research purposes and drug discovery.

The present invention also provides methods for drug discovery,genomics, proteomics, and pathway identification; comprising analyzingthe genomic or proteomic makeup of a MASC, coupled with analysis thereofvia bioinformatics, computer analysis via algorithms, to assemble andcompare new with known databases and compare and contract these. Thiswill identify key components, pathways, new genes and/or new patterns ofgene and protein expression and protein modification (proteomics) thatcould lead to the definition of targets for new compounds, antibodies,proteins, small molecule organic compounds, or other biologically activemolecules that would have therapeutic benefit.

EXAMPLES

The following examples are provided to illustrate but not limit theinvention.

Example 1 Selection, Culture and Characterization of Mouse MultipotentAdult Stem Cells (mMASC)

Cell Isolation and Expansion

All tissues were obtained according to guidelines from the University ofMinnesota IACUC. BM mononuclear cells (BMMNC) were obtained byficoll-hypaque separation of BM was obtained from 5-6 week old ROSA26mice or C57/BL6 mice. Alternatively, muscle and brain tissue wasobtained from 3-day old 129 mice. Muscles from the proximal parts offore and hind limbs were excised from and thoroughly minced. The tissuewas treated with 0.2% collagenase (Sigma Chemical Co, St Louis, Mo.) for1 hour at 37° C., followed by 0.1% trypsin (Invitrogen, Grand Island,N.Y.) for 45 minutes. Cells were then triturated vigorously and passedthrough a 70-um filter. Cell suspensions were collected and centrifugedfor 10 minutes at 1600 rpm. Brain tissues was dissected and mincedthoroughly. Cells were dissociated by incubation with 0.1% trypsin and0.1% DNAse (Sigma) for 30 minutes at 37° C. Cells were then trituratedvigorously and passed through a 70-um filter. Cell suspension wascollected and centrifuged for 10 minutes at 1600 rpm.

BMMNC or muscle or brain suspensions were plated at 1×10⁵/cm² inexpansion medium [2% FCS in low glucose Dulbecco's minimal essentialmedium (LG-DMEM), 10 ng/mL each platelet derived growth factor (PDGF),epidermal growth factor (EGF) and leukemia inhibitory factor (LIF)] andmaintained at 5×10³/cm². After 3-4 weeks, cells recovered bytrypsin/EDTA were depleted of CD45⁺/glycophorin (Gly)-A⁺ cells withmicromagnetic beads. Resulting CD45⁻/Gly-A⁻ cells were replated at 10cells/well in 96-well plates coated with FN and were expanded at celldensities between 0.5 and 1.5×10³/cm². The expansion potential of MASCwas similar regardless of the tissue from which they were derived (FIG.1).

Characterization of MASC

Phenotypically, mMASC derived from BM, muscle and brain and cultured onFN were CD13⁺, CD44⁻, CD45⁻, class-I and class-II histocompatibilityantigen⁻, Flk1^(low) and cKit⁻, identical to the characteristics ofhMASC, as described in Internation Application No. PCT/US00/21387.Although cell expansion during the initial 2-3 months was greater whencells were cultured on collagen type IV, laminin or Matrigel™, cells hadphenotypic characteristics of MSC, i.e., expressed CD44 and did notexpress CD13. As with human cells, mMASC cultured on FN expressedtranscripts for oct-4, and the LIF-R.

Approximately 1% of wells seeded with 10 CD45⁻GlyA⁻ cells yieldedcontinuous growing cultures. This suggests that the cells capable ofinitiating MASC cultures are rare and likely less that 1/1,000 ofCD45⁻/GlyA⁻ cells. mMASC cultured on FN were 8-10 μm in diameter with alarge nucleus and scant cytoplasm. Several populations have beencultured for >100 PDs. The morphology and phenotype of cells remainedunchanged throughout culture.

mMASC that had undergone 40 and 102 PDs were harvested and telomerelengths evaluated. Telomere length was measured using the TelomereLength Assay Kit from Pharmingen (New Jersey, USA) according to themanufacturer's recommendations. Average telomere length (ATL) of mMASCcultured for 40 PDs was 27 Kb. When re-tested after 102 PDs, ATLremained unchanged. For karyotyping of mMASC, cells were subcultured ata 1:2 dilution 12 h before harvesting, collected with trypsin-EDTA, andsubjected to a 1.5 h colcemid incubation followed by lysis withhypotonic KCl and fixation in acid/alcohol as previously described(Verfaillie et al., 1992). Cytogenic analysis was conducted on a monthlybasis and showed a normal karyotype, except for a single population thatbecame hyperdiploid after 45 PDs, which was no longer used for studies.

Murine MASC obtained after 46 to >80 PDs were tested by Quantitative(Q)-RT-PCR for expression levels of Oct4 and Rex1, two transcriptionfactors important in maintaining an undifferentiated status of ES cells.RNA was extracted from mouse MASC, neuroectodermal differentiatedprogeny (day 1-7 after addition of bFGF) and mouse ES cells. RNA wasreverse transcribed and the resulting cDNA underwent 40 rounds ofamplification (ABI PRISM 7700, Perkin Elmer/Applied Biosystems) with thefollowing reaction conditions: 40 cycles of a two step PCR (95° C. for15 seconds, 60° C. for 60 seconds) after initial denaturation (95° C.for 10 minutes) with 2 μl of DNA solution, 1× TaqMan SYBR GreenUniversal Mix PCR reaction buffer. Primers are listed in Table 1.

TABLE 1 Primers used NEO 5′-TGGATTGCACGCAGGTTCT-3′5′-TTCGCTTGGTGGTCGAATG-3′ Oct4 5′-GAAGCGTTTCTCCCTGGATT-3′5′-GTGTAGGATTGGGTGCGTT-3′ Rex 1 5′-GAAGCGTTCTCCCTGGAATTTC-3′5′-GTGTAGGATTGGGTGCGTTT-3′ otx 1 5′-GCTGTTCGCAAAGACTCGCTAC-3′5′-ATGGCTCTGGCACTGATACGGATG-3′ otx2 5′-CCATGACCTATACTCAGGCTTCAGG-3′5′-GAAGCTCCATATCCCTGGGTGGAAAG-3′ Nestin 5′ 5′-GGAGTGTCGCTTAGAGGTGC-3′5′-TCCAGAAAGCCAAGAGAAGC-3′

mRNA levels were normalized using GAPDH as housekeeping gene, andcompared with levels in mouse ES cells. Oct4 and Rex 1 mRNA were presentat similar levels in BM, muscle and brain derived MASC. Rex1 mRNA levelswere similar in mMASC and mES cells, while Oct4 mRNA levels were about1,000 fold lower in MASC than in ES cells.

Expressed Gene Profile of Mouse BM, Muscle and Brain Derived MASC isHighly Similar

To further evaluate whether MASC derived from different tissues weresimilar, the expressed gene profile of BM, muscle and brain derived MASCwas examined using U74A Affimetrix gene array. Briefly, mRNA wasextracted from 2-3×10⁶ BM, muscle or brain derived-MASC, cultured for 45population doublings. Preparation of cDNA, hybridization to the U74Aarray containing 6,000 murine genes and 6,000 EST clusters, and dataacquisition were done per manufacturer's recommendations (all fromAffimetrix, Santa Clara, Calif.). Data analysis was done using GeneChip®software (Affimetrix). Increased or decreased expression by a factor of2.2 fold (Iyer V. R. et al., 1999; Scherf U. et al., 2000; Alizadeh A.A. et al., 2000) was considered significant. r² value was determinedusing linear regression analysis (FIG. 2).

Comparison between the expressed gene profile in MASC from the threetissues showed that <1% of genes were expressed at >2.2-fold differentlevels in MASC from BM than muscle. Likewise, only <1% of genes wereexpressed >2.2-fold different level in BM than brain derived MASC. Asthe correlation coefficient between the different MASC populationswas >0.975, it was concluded that MASC derived from the differenttissues are highly homologous, in line with the phenotypic describedabove and the differentiation characteristics described in Example 5.

Using the mouse-specific culture conditions, mMASC cultures have beenmaintained for more than 100 cell doublings. mMASC cultures have beeninitiated with marrow from C57Bl/6 mice, ROSA26 mice and C57BL/6 micetransgenic for the -HMG-LacZ.

Example 2 Selection and Culture of Rat Multipotent Adult Stem Cells(rMASC)

BM and MNC from Sprague Dawley or Wistar rats were obtained and platedunder conditions similar for mMASC. After 21-28 days, cells weredepleted of CD45⁺ cells, and the resulting CD45⁻ cells were subculturedat 10 cells/well.

Similar to mMASC, rMASC have been culture expanded for >100 PDs.Expansion conditions of rat MASC culture required the addition of EGF,PDGF-BB and LIF and culture on FN, but not collagen type I, laminin orMatrigel™. rMASC were CD44, CD45 and MHC class I and II negative, andexpressed high levels of telomerase. The ability of a normal cell togrow over 100 cell doublings is unprecedented, unexpected and goesagainst conventional dogma of more than two decades.

Rat MASC that had undergone 42 PDs, 72 PDs, 80 PDs, and 100 PDs, wereharvested and telomere lengths evaluated. Telomeres did not shorten inculture, as was determined by Southern blot analysis after 42 PDs, 72PDs, 80 PDs, and 100 PDs. Monthly cytogenetic analysis of rat MASCrevealed normal karyotype.

Example 3 Selection and Culture of Human Multipotent Adult Stem Cells(hMASC)

BM was obtained from healthy volunteer donors (age 2-50 years) afterinformed consent using guidelines from the University of MinnesotaCommittee on the use of Human Subject in Research. BMMNC were obtainedby Ficoll-Paque density gradient centrifugation and depleted of CD45⁺and glycophorin-A⁺ cells using micromagnetic beads (Miltenyii Biotec,Sunnyvale, Calif.).

Expansion conditions: 5×10³ CD45⁻/GlyA⁻ cells were diluted in 200 μLexpansion medium [58% DMEM-LG, 40% MCDB-201 (Sigma Chemical Co, StLouis, Mo.), supplemented with 1× insulin-transferrin-selenium (ITS), 1×linoleic-acid bovine serum albumin (LA-BSA), 10⁻⁸ M Dexamethasone, 10⁻⁴M ascorbic acid 2-phosphate (all from Sigma), 100 U penicillin and 1,000U streptomycin (Gibco)] and 0-10% fetal calf serum (FCS) (HycloneLaboratories, Logan, Utah) with 10 ng/ml of EGF (Sigma) and 10 ng/mlPDGF-BB (R&D Systems, Minneapolis, Minn.)] and plated in wells of 96well plates that had been coated with 5 ng/ml of FN (Sigma). Medium wasexchanged every 4-6 days. Once wells were >40-50% confluent, adherentcells were detached with 0.25% trypsin-EDTA (Sigma) and replated at 1:4dilution in MASC expansion medium and bigger culture vessels coated with5 ng/ml FN to maintain cell densities between 2 and 8×10³ cells/cm².

Undifferentiated MASC did not express CD31, CD34, CD36, CD44, CD45,CD62-E, CD62-L, CD62-P, HLA-class I and II, cKit, Tie, Tek, α_(v)β₃,VE-cadherin, vascular cell adhesion molecule (VCAM), intracellularadhesion molecule (ICAM)-1. MASC expressed low/very low levels ofβ2-microglobulin, α_(v)β₅, CDw90, AC133, Flk1 and Flt1, and high levelsof CD13 and CD49b (FIG. 3).

Example 4 Immunophenotypic Analysis

Immunofluorescence

1. Cultured cells were fixed with 4% paraformaldehyde and methanol atroom temperature, and incubated sequentially for 30 min each withprimary antibody, and with or without secondary antibody. Between steps,slides were washed with PBS/BSA. Cells were examined by fluorescencemicroscopy (Zeiss Axiovert; Carl Zeiss, Inc., Thornwood, N.Y.) andconfocal fluorescence microscopy (Confocal 1024 microscope; OlympusAX70, Olympus Optical Co. LTD, Japan). To assess the frequency ofdifferent cell types in a given culture, the number of cells werecounted that stained positive with a given antibody in four visualfields (50-200 cells per field).

2. Harvested tissues: Cytospin specimens of blood and BM were fixed withacetone (Fisher Chemicals) for 10 min at room temperature. For solidorgans, 5 μm thick fresh frozen sections of tissues were mounted onglass slides and immediately fixed in acetone for 10 min at roomtemperature. Following incubation with isotype sera for 20 min, cytospinpreparations or tissue sections were serially stained for tissuespecific antigens, β-gal and a nuclear counter stain (DAPI or TO-PRO-3).Cover slips were mounted using Slowfade-antifade kit (Molecular ProbesInc., Eugene, Oreg., USA). Slides were examined by fluorescencemicroscopy and confocal fluorescence microscopy.

3. Antibodies: Cells were fixed with 4% paraformaldehyde at roomtemperature or methanol at −20° C., and incubated sequentially for 30min each with primary Ab, and FITC or Cy3 coupled anti-mouse- oranti-rabbit-IgG Ab. Between each step slides were washed with PBS+1%BSA. PE or FITC-coupled anti-CD45, anti-CD31, anti-CD62E, anti-Mac1,anti-CD19, anti-CD3, and anti-Teri 19 antibodies were obtained from BDPharmingen. Abs against GFAP (clone G-A-5, 1:400), galactocerebroside(GalC) (polyclonal, 1:50), MBP (polyclonal, 1:50), GABA (clone GB-69,1:100), parvalbumin (clone PARV-19, 1:2000), TuJ1 (clone SDL3D10,1:400), NF-68 (clone NR4, 1:400), NF-160 (clone NN 18, 1:40), and NF-200(clone N52, 1:400), NSE (polyclonal, 1:50), MAP2-AB (clone AP20, 1:400),Tau (polyclonal, 1:400), TH (clone TH-2, 1:1000), DDC (clone DDC-109,1:100), TrH (clone WH-3, 1:1000), serotonin (polyclonal, 1:2000),glutamate (clone GLU-4, 1:400), fast twitch myosin (clone MY-32; 1:400dilution) were from Sigma. DAPI and TOPRO-3 were from Molecular Probes.Abs against vWF (polyclonal; 1:50) Neuro-D (polyclonal, 1:50), c-ret(polyclonal, 1:50) and NurrI (polyclonal, 1:50) were from Santa CruzBiotechnology Inc., Santa Cruz, Calif. Abs against PSA-NCAM (polyclonal,1:500) from Phanmingen, San Diego, Calif. and against serotonintransporter (clone MAB 1564, 1:400), DTP (polyclonal, 1:200), Na-gatedvoltage channel (polyclonal, 1:100), glutamate-receptors-5, -6 and -7(clone 3711:500) and NMDA (polyclonal 1:400) receptor from ChemiconInternational, Temecula, Calif. Anti-nestin (1:400) Abs were a kind giftfrom Dr. U. Lendahl, University of Lund, Sweden. Antibodies against NSE(1:50) pan-cytokeratin (catalog number C-2562; 1:100), CK-18 (C-8541;1:300), albumin (A-6684; 1:100) were all obtained from Sigma. Polyclonalantibodies against Flk1, Flt1, Tek, HNF-1β were obtained from Santa CruzBiotechnology Inc., Santa Cruz, Calif. Anti-nestin (1:400) antibodieswere a kind gift from Dr. U. Lendahl, University of Lund, Sweden.Control-mouse, -rabbit or, -rat IgGs and FITC/PE/Cy3- and Cy5-labeledsecondary antibodies were obtained from Sigma. Rabbit anti-β-gal-FITCantibody was obtained from Rockland Immunochemicals, USA. TO-PRO-3 wasobtained from Molecular Probes Inc. and DAPI was obtained from Sigma.

B. X-GAL staining: Tissue sections were stained by for β-galactosidaseenzyme activity using β-gal staining kit from Invitrogen, pH 7.4.Manufacturer's instructions were followed except for the fixation step,during which the tissue sections were incubated for 5 min instead of 10min.C. FACS: For FACS, undifferentiated MASC were detached and stainedsequentially with anti-CD44, CD45, CD13, cKit, MHC-class I and II, orb2-microglobulin (BD Pharmingen) and secondary FITC or PE coupledantibodies, fixed with 2% paraformaldehyde until analysis using aFACS-Calibur (Becton-Dickinson).

Example 5 Single Cell Origin of Differentiated Lineages from MASC

The differentiation ability of mMASC or rMASC was tested by addingdifferentiation factors (cytokines) chosen based on what has beendescribed for differentiation of hMASC or ES cells to mesoderm,neuroectoderm, and endoderm. Differentiation required that cells werereplated at 1-2×10⁴ cells/cm² in serum free medium, without EGF, PDGF-BBand LIF, but with lineage specific cytokines. Differentiation wasdetermined by immunohistology for tissue specific markers [slow twitchmyosin and MyoD (muscle), von-Willebrand factor (vWF) and Tek(endothelium), NF200 and MAP2 (neuroectodermal), and cytokeratin-18 andalbumin (endodermal)], RT-PCR, and functional studies.

MASC Differentiation into Neuroectodermal Cells

Palmer et al. showed that neuroprogenitors can be culture expanded withPDGF-BB and induced to differentiate by removal of PDGF and addition ofbFGF as a differentiation factor. Based on those studies and studiesconducted using hMASC, mMASC and rMASC were plated in FN coated wellswithout PDGF-BB and EGF but with 100 ng/mL bFGF. Progressive maturationof neuron-like cells was seen throughout culture. After 7 days, themajority of cells expressed nestin. After 14 days, 15-20% of MASCacquired morphologic and phenotypic characteristics of astrocytes(GFAP⁺), 15-20% of oligodendrocytes (galactocerebroside (GalC)⁺) and50-60% of neurons (neurofilament-200 (NF-200)⁺). NF200, GFAP or GalCwere never found in the same cell, suggesting that it is unlikely thatneuron-like cells were hMASC or glial cells that inappropriatelyexpressed neuronal-markers. Neuron-like cells also expressed Tau, MAP2and NSE. Approximately 50% of neurons expressed gamma-amino-butyric-acid(GABA) and parvalbumin, 30% tyrosine hydroxylase and dopa-decarboxylase(DDC), and 20% serotonin and tryptophan hydroxylase. Differentiation wassimilar when MASC had been expanded for 40 or >90 PDs. Q-RT-PCR,performed as described in Example 1, confirmed expression ofneuroectodermal markers: on day 2 MASC expressed otx1 and otx2 mRNA, andafter 7 days nestin mRNA was detected.

The effect of fibroblast growth factor (FGF)-8b as a differentiationfactor was tested next. This is important in vivo for midbraindevelopment and used in vitro to induce dopaminergic and serotoninergicneurons from murine ES cells on hMASC. When confluent hMASC (n=8) werecultured with 10 ng/mL FGF-8b+EGF, differentiation into cells stainingpositive for neuronal markers but not oligodendrocytes and astrocyteswas seen. Neurons had characteristics of GABAergic (GABA⁺; 40±4%),dopaminergic (DOPA, TH, DCC and DTP⁺, 26±5%) and serotoninergic (TrH;serotonin and serotonin-transporter⁺, 34±6%) neurons. DOPA⁺ neuronsstained with Abs against Nurr1 suggesting differentiation to midbrain DAneurons. FGF-8b induced neurons did not have electrophysiologicalcharacteristics of mature neurons. Therefore, cocultured cells from3-week old FGF-8b supported cultures with the glioblastoma cell line,U-87, and FGF-8b for an additional 2-3 weeks.

Neurons acquired a more mature morphology with increased cell size andnumber, length and complexity of the neurites, and acquiredelectrophysiological characteristics of mature neurons (a transientinward current, blocked reversibly by 1 μM tetrodotoxin (TTX) togetherwith the transient time course and the voltage-dependent activation ofthe inward current is typical for voltage-activated sodium currents,found only in mature neurons).

When hMASC (n=13) were cultured with 10 ng/m brain-derived neurotrophicfactor (BDNF)+EGF, differentiation was to exclusively DOPA, TH, DCC, DTPand Nurr1 positive neurons. Although BDNF supports neuraldifferentiation from ES cells and NSC (Peault, 1996; Choi et al. 1998),no studies have shown exclusive differentiation to DA-like neurons.

Similar results were seen for mMASC induced with bFGF and rMASC withbFGF and BDNF. Further studies on MASC-derived neuronal cells arepresented in Example 10.

MASC Differentiation into Endothelial Cells

As an example of mesoderm, differentiation was induced to endothelium.Undifferentiated mMASC or rMASC did not express the endothelial markersCD31, CD62E, Tek or vWF, but expressed low levels of Flk1. mMASC orrMASC were cultured in FN-coated wells with 10 ng/mL of the endothelialdifferentiation factor VEGF-B. Following treatment with VEGF for 14days, >90% of MASC, irrespective of the number of PDs they hadundergone, expressed Flt1, CD31, vWF or CD62, consistent withendothelial differentiation. Like primary endothelial cells,MASC-derived endothelial cells formed vascular tubes within 6 hoursafter replating in Matrigel™.

Similarly, hMASC express Flk1 and Flt1 but not CD34, Muc18 (P1H12),PECAM, E- and P-selectin, CD36, or Tie/Tek. When hMASC 2×10⁴ cells/cm²were cultured in serum free medium with 20 ng/mL vascular endothelialgrowth factor (VEGF), cells expressed CD34, VE-cadherin, VCAM and Muc-18from day 7 on. On day 14, they also expressed Tie, Tek, Flk1 and Flt1,PECAM, P-selectin and E-selectin, CD36, vWF, and connexin-40.Furthermore, cells could uptake low-density lipoproteins (LDL). Resultsfrom the histochemical staining were confirmed by Western blot. Toinduce vascular tube formation, MASC cultured for 14 days with VEGF werereplated on Matrigel™ with 10 ng/mL VEGF-B for 6 h. Endothelialdifferentiation was not seen when hMASC cultured in >2% FCS were used.In addition, when FCS was left in the media during differentiation, noendothelial cells were generated.

At least 1000-fold expansion was obtained when hMASC were sub-cultured,suggesting that endothelial precursors generated from hMASC continue tohave significant proliferative potential. Cell expansion was evengreater when FCS was added to the cultures after day 7.

When hMASC derived endothelial cells were administered intravenously(I.V.) in NOD-SCI mice who have a human colon-carcinoma implanted underthe skin, contribution of the human endothelial cells could be seen tothe neovascularization in the tumors. It may therefore be possible toincorporate genetically modified endothelial cells to derive atherapeutic benefit, i.e., to inhibit angiogenesis in for example canceror to promote it to enhance vascularization in limbs or other organssuch as the heart. Further studies on MASC-derived endothelial cells arepresented in Example 9.

MASC Differentiation into Endoderm

Whether mMASC or rMASC could differentiate to endodermal cells wastested. A number of different culture conditions were tested includingculture with the diffentiation factors keratinocyte growth factor (KGF),hepatocyte growth factor (HGF) and FGF-4, either on laminin, collagen,FN or Matrigel™ coated wells. When re-plated on Matrigel™ with 10 ng/mLFGF4+10 ng/mL HGF, approximately 70% of MASC acquired morphologic andphenotypic characteristics of hepatocyte-like cells. Cells becameepithelioid, approximately 10% of cells became binucleated, and about70% of cells stained positive for albumin, cytokeratin (CK)-18, andHNF-1P.

Endodermal-like cells generated in FGF4 and HGF containing cultures alsohad functional characteristics of hepatocytes, determined by measuringurea levels in supernatants of undifferentiated MASC and FGF4 andHGF-induced MASC using the Sigma Urea Nitrogen Kit 640 according to themanufacturer's recommendations. No urea was detected in undifferentiatedMASC cultures. Urea production was 10 μg/cell/hr 14 days after addingFGF4 and HGF and remained detectable at similar levels until day 25.This is comparable to primary rat hepatocytes grown in monolayer.Presence of albumin together with urea production supports the notion ofhepatic differentiation from MASC in vitro. Further studies onMASC-derived hepatocytes are presented in Example 11.

Given the likely existence of an endodermal lineage precursor cell, MASClikely give rise to a cell that forms various cells in the liver in thepancreas both exocrine and endocrine components and other endodermalderived cell tissue lineages.

MASC derived from muscle or brain were induced to differentiate tomesoderm (endothelial cells), neuroectoderm (astrocytes and neurons) andendoderm (hepatocyte-like cells) using the methods described above forBM-derived MASC.

Transduction

To demonstrate that differentiated cells were single cell derived andMASC are indeed “clonal” multipotent cells, cultures were made in whichMASC had been transduced with a retroviral vector and undifferentiatedcells and their progeny were found to have the retrovirus inserted inthe same site in the genome.

Studies were done using two independently derived ROSA26 MASC, twoC57BL/6 MASC and one rMASC population expanded for 40 to >90PDs, as wellas with the eGFP transduced “clonal” mouse and “clonal” rMASC. Nodifferences were seen between eGFP transduced and untransduced cells. Ofnote, eGFP expression persisted in differentiated MASC.

Specifically, murine and rat BMMNC cultured on FN with EGF, PDGF-BB andLIF for three weeks were transduced on two sequential days with an eGFPoncoretroviral vector. Afterwards, CD45⁺ and GlyA⁺ cells were depletedand cells sub-cultured at 10 cells/well. eGFP-transduced rat BMMNC wereexpanded for 85 PDs. Alternatively, mouse MASC expanded for 80 PDS wereused. Subcultures of undifferentiated MASC were generated by plating 100MASC from cultures maintained for 75 PDs and re-expanding them to >5×10⁶cells. Expanded MASC were induced to differentiate in vitro toendothelium, neuroectoderm and endoderm. Lineage differentiation wasshown by staining with antibodies specific for these cell types, asdescribed in Example 4.

Single Cell Origin of Mesodermal and Neuroectodermal Progeny

To prove single cell origin of mesodermal and neuroectodermaldifferentiated progeny retroviral marking was used (Jordan et al., 1990;Nolta et al., 1996). A fraction of hMASC obtained after 20 PDs wastransduced with an MFG-eGFP retrovirus. eGFP⁺ hMASC were diluted innon-transduced MASC from the same donors to obtain a final concentrationof ˜5% transduced cells. These mixtures were plated at 100 cells/welland culture expanded until >2×10⁷ cells were obtained. 5×10⁶ MASC eachwere induced to differentiate to skeletal myoblasts, endothelium andneuroectodermal lineages. After 14 days under differentiationconditions, cells were harvested and used to identify the retroviralintegration site and co-expression of eGFP and neuroectodermal, muscleand endothelial markers.

For myoblast differentiation, hMASC were plated at 2×10⁴ cells/cm² in 2%FCS, EGF and PDGF containing expansion medium and treated with 3 μM5-azacytidine in the same medium for 24 h. Afterwards, cells weremaintained in expansion medium with 2% FCS, EGF and PDGF-BB. Forendothelial differentiation, hMASC were replated at 2×10⁴ cells/cm² inserum-free expansion medium without EGF and PDGF but with 10 ng/mlVEGF-B for 14 days.

Immunofluorescence evaluation showed that 5-10% of cells in culturesinduced to differentiate with 5-azacytidine stained positive for eGFPand skeletal actin, 5-10% of cells induced to differentiate toendothelium costained for eGFP and vWF, and 5-10% of cells induced todifferentiate to neuroectoderm costained for eGFP and either NF-200,GFAP or MBP. To define the retroviral insertion site, the host genomicflanking region in MASC and differentiated progeny was sequenced. Thenumber of retroviral inserts in the different populations was betweenone and seven. As shown in Table 2, a single, identical sequenceflanking the retroviral insert in muscle, endothelium andneuroectodermal cells in population A16 that mapped to chromosome 7 wasidentified.

TABLE 2 Single cell origin of endothelium, muscle andneuroectodermal cells Sequence: 3′-LTR-ccaaatt Clone A16TAG CGGCCGCTTG AATTCGAACG CGAGACTACT (Chrom. 7) GTGACTCACA CT 5-TAG CGGCCGCTTG AATTCGAACG CGAGACTACT Azacytidine GTGACTCACA CT VEGFTAG CGGCCGCTTG AATTCGAACG CGAGACTACT GTGACTCACA CT bFGFTAG CGGCCGCTTG AATTCGAACG CGAGACTACT GTGACTCACA CT Clone A12-ATTTATA TTCTAGTTTAT TTGTGTTTGGG A (Chrom. GCAGACGAGG 9) 5-ATTTATA TTCTAGTTTAT TTGTGTTTGGG Azacytidine GCAGACGAGG VEGFATTTATA TTCTAGTTTAT TTGTGTTTGGG GCAGACGAGG bFGFATTTATA TTCTAGTTTAT TTGTGTTTGGG GCAGACGAGG Clone A12-TCCTGTCTCA TTCAAGCCAC ATCAGTTACA A (Chrom. TCTGCATTTT 12) 5-TCCTGTCTCA TTCAAGCCAC ATCAGTTACA Azacytidine TCTGCATTTT VEGFTCCTGTCTCA TTCAAGCCAC ATCAGTTACA TCTGCATTTT bFGFTCCTGTCTCA TTCAAGCCAC ATCAGTTACA TCTGCATTTT

Primers specific for the 3′ LTR were designed and for the flankinggenomic sequence are shown in Table 3 and using Real-time PCR, it wasconfirmed that the retroviral insert site was identical inundifferentiated and differentiated cells. These results proved that theflanking sequence and the eGFP DNA sequence was present in similarquantities. Clone A12 contained two retroviral inserts, located onchromosome 1 and 7 respectively, and both flanking sequences could bedetected not only in hMASC but also muscle, endothelium andneuroectodermal lineages. To determine whether this represented progenyof a single cell with two retroviral integrants or progeny of two cells,Real-Time PCR was used to compare the relative amount of the chromosome1 and 7 flanking sequence to eGFP. It was found that similar amounts ofboth flanking regions were present in hMASC, muscle, endothelium andneuroectodermal cells, suggesting that a single cell with two retroviralinserts was likely responsible for the eGFP positive hMASC anddifferentiated progeny. In the other populations containing 3 or moreretroviral inserts we were not able to determine whether the insertswere due to multiple insertion sites in a single cells or multiple cellscontributing to the eGFP positive fraction. Nevertheless, our findingthat in 2 populations, progeny differentiated into muscle, endotheliumand neuroectoderm are derived from a single BM derived progenitor celldefinitively proves for the first time that primitive cells can becultured from BM that differentiate at the single cell level in cells ofmesodermal lineage as well as the three different lineages of theneuroectoderm.

TABLE 3 Flanking regions and primers Clone Genomic sequence RatGATCCTTTGGGAGGGTCTCCT CAGATTGATTGACTGCCCA flankingCCTCGGGGGTCTTTCAAAGTAACTCCAAAAGAAGAATGGG sequenceTTGTTAGTTATTAAACGGTTCTTAGTAAAGTTTTGGTTTT GGGAATCACAGTAACAACTCACATCACAACTCCAATCGTT CCGTGAAA Mouse GATCCTTGGGAGGGTCTCCT CAGATTGATTGACTGCCCATflanking AAGTTATAAGCTGGCATGACTGTGT TGCTAAGGACACTGG sequence TGAAAGC

Bold: MSCV LTR; Bold and underlined: MSCV LTR primer used for Q-PCRItalics and underlined: Flanking sequence primers used for Q-PCR.

Example 6 Homing and Engraftment of Mammalian MASC into Numerous Organsin the Body

mMASC were tested to determine whether they had the ability to engraftand differentiate in vivo into tissue specific cells. mMASC were grownas described in Example 1 from a LacZ transgenic C57 Black 6, ROSA 26mouse. 10⁶ mMASC from the LacZ mouse were I.V. injected into NOD-SCIDmice tail veins with or without 250 Rads of total body radiation 4-6 hrsprior to the injection. The animals were sacrificed by cervicaldislocation at 4-24 weeks after the injections.

Tissue Harvest

Blood and bone marrow: 0.5-1 ml of blood was obtained at the timeanimals were sacrificed. BM was collected by flushing femurs and tibias.For phenotyping, red cells in blood and BM were depleted using ice coldammonium chloride (Stem Cell Technologies Inc., Vancouver, Canada) and10⁵ cells used for cytospin centrifugation. For serial transplantation,5×10⁷ cells from 2 femurs and 2 tibias were transplanted into individualsecondary recipients via tail vein injection. Secondary recipients weresacrificed after 7-10 weeks.

Solid organs: Lungs were inflated with 1 ml 1:4 dilution of OCT compound(Sakura-Finetek Inc, USA) in PBS. Specimens of spleen, liver, lung,intestine, skeletal muscle, myocardium, kidney and brain of therecipient animals were harvested and cryopreserved in OCT at −80° C. andin RNA Later (Ambion Inc., Austin, Tex., USA) at −20° C. forquantitative PCR.

mMASC Engraft and Differentiate in Tissue specific Cells In Vivo

Engraftment of the β-gal/neomycin (NEO) transgene-containing cells(Zambrowicz et al., 1997) was tested by immunohistochemistry for β-galand by Q-PCR for NEO. Immunohistochemistry and Q-PCR were performed asdescribed in Examples 5 and 1 respectively. Primers are listed in Table1.

Engraftment, defined as detection of >1% anti-β-gal cells, was seen inhematopoietic tissues (blood, BM and spleen) as well as epithelium oflung, liver, and intestine of all recipient animals as shown in Table 4and FIG. 4.

TABLE 4 Engraftment levels in NOD-SCID mice transplanted with ROSA26MASC. Engraftment levels (%) determined by Time immunofluorescence or(Q-PCR) Animal (Weeks) Radiation Marrow Blood Spleen Liver LungIntestine 1 4 No 2 (1) 2 5 7 4 2 2 5 No 3 (4) 4 5 9 5 3 3 10 No 1 3 3 63 2 4 16 No 4 2 3 4 3 4 (4.9) 5 24 No 3 2 3 6 4 1 6 8 Yes 8 (8) 6 4 5 2(1.1) 7 7 8 yes 10  8 7 (7.3)  4 6 8 8 8 Yes 5 8 3 5 5 6 9 8 Yes 7 5 5 64 6 10 10 Yes 5 (6) 7 9 (12.5) 5 2 8 11 11 Yes 8 8 6 5 3 10 (11.9) 12 11Yes 6 5 4 8 (6.2) 10 (12.3) 8 SR-1 7 Yes 6 7 5 1 (1.7) 5 8 SR-2 10 Yes 54 8 3 4 6

β-gal⁺ cells in BM (FIG. 4B-F) and spleen (FIG. 4H-I) co-labeled withanti-CD 45, anti-CD19, anti-Mac1, anti-Gr1 and anti-TER119 Abs. Similarresults were seen for peripheral blood. Of note, no β-gal⁺CD3⁺ T cellswere seen in either blood, BM or spleen even though β-gal⁺CD3⁺ T-cellswere seen in chimeric mice. The reason for this is currently not known.

Engraftment in the spleen occurred mostly as clusters of donor cells,consistent with the hypothesis that when MASC home to the spleen, theyproliferate locally and differentiate to form a colony of donor cells,similar to CFU-S. It is not believed that differentiation of mMASC intohematopoietic cells in vivo can by attributed to contamination of themMASC with HSC. First, BMMNC are depleted of CD45 cells by columnselection before mMASC cultures are initiated. Second, early mesodermalor hematopoietic transcription factors, including brachyury (Robertsonet al., 2000), GATA-2 and GATA-1 (Weiss et al., 1995), are not expressedin undifferentiated mMASC, as shown by cDNA array analysis. Third, theculture conditions used for mMASC are not supportive for HCS. Fourth allattempts at inducing hematopoietic differentiation from hMASC in vitro,by co-culturing hMASC with hematopoietic supportive feeders andcytokines, have been unsuccessful (Reyes et al., 2001).

Significant levels of mMASC engraftment were also seen in liver,intestine and lung. Triple-color immunohistochemistry was used toidentify epithelial (CK⁺) and hematopoietic (CD45⁺) cells in the sametissue sections of the liver, intestine and lung. In the liver, β-gal⁺donor-derived cells formed cords of hepatocytes (CK18⁺CD45⁺ oralbumin⁺), occupying about 5-10% of a given 5 μm section (FIG. 4K-M).Several CK18⁺CD45⁺β-gal⁻ hematopoietic cells of recipient origin weredistinctly identified from the epithelial cells. Albumin⁺β-gal⁺ andCK18⁺β-gal⁺ cells engrafted in cords of hepatocytes surrounding portaltracts, a pattern seen in hepatic regeneration from hepatic stem cellsand oval cells (Alison et al., 1998; Petersen et al., 1999). This andthe fact that only 5/20 sections contained donor cells, is consistentwith the notion that stem cells engraft in some but not all areas of theliver, where they proliferate and differentiate into hepatocytes.

Engraftment in the intestine was also consistent with what is knownabout intestinal epithelial stem cells. In the gut, each crypt containsa population of 4-5 long-lived stem cells (Potten, 1998). Progeny ofthese stem cells undergo several rounds of division in the middle andupper portions of cypts and give rise to epithelial cells that migrateupwards, out of the crypt, onto adjacent villi. Donor derived,β-gal⁺panCK⁺CD45⁻ epithelial cells entirely covered several villi (FIG.4O-P). In some villi, β-gal⁺panCK⁺CD45⁻ cells constituted only 50% ofthe circumference (solid arrows, FIG. 4P) suggesting engraftment in onebut not both crypts. Several β-gal⁺panCK⁻ cells were distinctly seen inthe core of intestinal villi (open arrow, FIG. 4O). These cellsco-stained for CD45 (FIG. 4P), indicating that they were donor-derivedhematopoietic cells. In the lung, the majority of donor cells gave riseto β-gal⁺panCK⁺CD45⁻ alveolar epithelial cells whereas, mosthematopoietic cells were of recipient-origin (panCK⁻CD45⁺β-gal⁻) (FIG.4R).

Levels of engraftment detected by immunohistochemistry were concordantwith levels determined by Q-PCR for NEO (Table 4). Engraftment levelswere similar in animals analyzed after 4 to 24 weeks following I.V.injection of MASC (Table 4).

No contribution was seen to skeletal or cardiac muscle. In contrast toepithelial tissues and the hematopoietic system, little to no cellturnover is seen in skeletal or cardiac muscle in the absence of tissueinjury. Therefore, one may not expect significant contribution of stemcells to these tissues. However, engraftment was not found in skin andkidney, two organs in which epithelial cells undergo rapid turnover. Itis shown in the blastocyst injection experiments (Example 8) that mMASCcan differentiate into these cell types; one possible explanation forthe lack of engraftment in these organs in post-natal recipients is thatin MASC do not home to these organs, a hypothesis that is currentlybeing evaluated. Although mMASC differentiated into neuroectoderm-likecells ex vivo, no significant engraftment of mMASC was seen in thebrain, and rare donor cells found in the brain did not co-label withneuroectodermal markers. Two recent publications demonstrated that donorderived cells with neuroectodermal characteristics can be detected inthe brain of animals that underwent BM transplantation. However, a fullyablative preparative regimen prior to transplantation or transplantationin newborn animals was used, conditions associated with break-down ofthe blood-brain barrier. Cells were infused in non-irradiated adultanimals, or animals treated with low dose radiation, where theblood-brain barrier is intact or only minimally damaged. This mayexplain the lack of mMASC engraftment in the CNS.

Confluent MASCdo not Differentiate In Vivo

As control, ROSA26-MASC were infused and grown to confluence priorinjection. MASC allowed to become confluent lose their ability todifferentiate ex vivo in cells outside of the mesoderm, and behave likeclassical MSC (Reyes, M. et al. 2001). Infusion of 10⁶ confluent mMASCdid not yield significant levels of donor cell engraftment. Although fewβ-gal⁺ cells were seen in BM, these cells did not co-label withanti-CD45 Abs, indicating that MSC may engraft in tissues, but are nolonger able to differentiate into tissue specific cells in response tolocal cues.

MASC Derived Cells in Bone Marrow of Mice can be Serially Transferred

BM from mouse engrafted with ROSA26 MASC was tested to determine whetherthey contained cells that would engraft in secondary recipients. 1.5×10⁷BM cells, recovered from primary recipients 11 weeks after I.V. infusionof mMASC, were transferred to secondary irradiated NOD-SCID recipients(Table 4: animal SR-1 and SR-2). After 7 and 10 weeks, secondaryrecipients were sacrificed, and blood, BM, spleen, liver, lung andintestines of the recipient animal were analyzed for engraftment ofROSA26 donor cells by immunohistochemistry and Q-PCR for the NEO gene. Asimilar pattern of engraftment was seen in secondary recipients as inthe primary recipients. Four-8% of BM, spleen and PB cells wereβ-gal⁺CD45⁺; six and 8% of intestinal epithelial cells wereβ-gal⁺pan-CK⁺, and 4 and 5% of lung epithelial cells were β-gal⁺pan-CK⁺.Levels of engraftment in the liver of secondary recipients were lowerthan in the primary recipients (1 and 3% vs. 5 and 8% β-gal⁺CK18⁺). Thissuggests that mMASC may persist in the BM of the primary recipient anddifferentiate into hematopoietic cells as well as epithelial cells whentransferred to a second recipient.

MASC derived cells can produce insulin in vivo. MASC from ROSA26 micewere injected into irradiated NOD-SCID mice as described herein. Theresulting MASC derived cells co-stain for LacZ and insulin in a model ofstreptozotocin-induced diabetes.

Summary

One of the critical questions in “stem cell plasticity” is whether theengrafted and differentiated donor mMASC are functional. The resultsdescribed herein show that one animal developed a lymphoma in thymus andspleen after 16 weeks, as is commonly see in aging NOD-SCID mice(Prochazka et al., 1992). Phenotypic analysis showed that this B-celllymphoma was host-derived: CD19⁺ cells were β-gal⁻. Approximately 40% ofCD45⁻vWF⁺ cells in the vasculasture of the tumor stained with anti-β-galAbs, indicating that neoangiogenesis in the tumor was in part derivedfrom donor mMASC (FIG. 4T). This suggests that MASC give rise tofunctioning progeny in vivo. Likewise, higher levels of mMASCengraftment and differentiation in radiosensitive organs, such as thehematopoietic system and intestinal epithelium (Table 4, p<0.001),following low dose irradiation suggests that mMASC may contributefunctionally to host tissues.

These results showed that mammalian MASC can be purified, expanded exvivo, and infused I.V., homed to various sites in the body, engraft intonumerous organs, and that the cells are alive in these various organsone month or longer. Such donor cells, undifferentiated, anddifferentiated progeny are found, by virtue of the fluorescent marker,in organs including; but not limited to, the BM, spleen, liver and lung.These cells can be used to repopulate one or more compartment(s) toaugment or restore cell or organ function.

Example 7 Demonstration of In Vitro Hematopoiesis and Erythropoiesis

MASC from, human BM differentiate at the single cell level intoneuroectodermal, endodermal and many mesodermal lineages, includingendothelial cells. Because endothelium and blood are very closelyrelated in ontogeny, it can be hypothesized that MASC can differentiateinto hematopoietic cells. eGFP transduced human MASC, that are GlyA,CD45 and CD34 negative (n=20), were cocultured with the mouse yolk sacmesodermal cell line, YSM5, as suspension cell aggregates for 6 days inserum free medium supplemented with 10 ng/mL bFGF and VEGF. After sixdays, only eGFP⁺ cells. (i.e., MASC progeny) remained and YSM5 cells haddied.

Remaining cells were transferred to methylcellulose cultures containing10% fetal calf serum supplemented with 10 ng/mL bone morphogenic protein(BMP)4, VEGF, bFGF, stem cell factor (SCF), Flt3L, hyper IL6,thrombopoietin (TPO), and erythropoietin (EPO) for 2 weeks. In thesecultures, both adherent eGFP⁺ cells and small, round non-adherent cells,which formed many colonies attached to the adherent cells were detected.The non-adherent and adherent fractions were collected separately andcultured in 10% FCS containing medium with 10 ng/mL VEGF and bFGF for 7days. Adherent cells stained positive for vWF, formed vascular tubeswhen plated on ECM, and were able to uptake a-LDL, indicating theirendothelial nature. 5-50% of the non-adherent cells stained positive forhuman specific GlyA and HLA-class I by flow cytometry.Gly-A⁺/HLA-class-I⁺ cells were selected by FACS. On Wright-Giemsa, thesecells exhibited the characteristic morphology and staining pattern ofprimitive erythroblasts. Cells were benzidine and human Hb⁺ byimmunoperoxidase. By RT-PCR these cells expressed human specific Hb-e,but not Hb-a.

When replated in methylcellulose assay with 20% FCS and EPO, smallerythroid colonies were seen after 10 days, and 100% of these coloniesstained positive for human specific GlyA and Hb. As selection of MASCdepends on the depletion of CD45⁺ and Gly A⁺ cells from BM, and culturedMASC are CD45⁻ and GlyA⁻ at all times examined using both FACS and cDNAarray analysis, contamination of MASC with hematopoietic cells is veryunlikely.

Example 8 In Vivo Proof of the Multipotent Nature of MASC as Shown byMultiple Organ Chimerism Following Blastocyst Injections of the Cells

Important for therapeutic applications of these cells is the ability ofMASC to proliferate and differentiate into the appropriate cell types invivo. Up until this point the only cells that should be capable ofcontributing to the full constellation of tissues and organs in the bodyare ES cells. In order to analyze whether MASC could show the fullcapability of ES cells, they were assayed to determine theircontribution to the formation of various tissues by introducing theminto the early blastocyst and observing the fate of their progeny.

MASC were generated from marrow of ROSA26 mice that are transgenic forthe β-galactosidase (β-gal) gene (Rafii, S., et al. 1994, Blood.84:10-13) and expanded as described in Example 1. One or 10-12 ROSA26MASC obtained after 55-65 PDs were microinjected into 88 and 40 3.5-dayC57BL/6 blastocysts, respectively. Blastocysts (8/mother) weretransferred to 16 foster mothers and mice allowed to develop and be bornas shown in Table 5.

TABLE 5 Degree of chimerism following MASC injection in blastocyst MASC/Litters Total # NEO positive by Q-PCR blastocyst born pups born 0% 1-10%10-20% 20-40% >40% 10-12 4/11 22 5/22 13/22 2/22 1/22 1/22 (23%) (59%)(9%) (4.5%) (4.5%) 1 3/5  15 8/15  5/15 0/15 0/15 2/15 (53%) (33%) (0%)  (0%)  (13%)

Seven litters were born, in line with the birth rate seen in otherstudies during this period. The number of mice per litter varied between1 and 8, for a total of 37 mice. Animals born from microinjectedblastocysts were of similar size as normal animals and did not displayany overt abnormalities.

After four weeks, animals were evaluated for chimerism by clipping theirtails and assessing the contribution of β-gal/NEO transgene containingcells to the tails by Q-PCR for NEO. Percent chimerism was determined bycomparing the number of NEO copies in test samples with that in tissuefrom ROSA26 mice according to manufacturer's recommendations (7700 ABIPRISM Detector Software 1.6). Chimerism could be detected in 70% of micederived from blastocysts in which 10 to 12 MASC had been injected and50% of mice derived from blastocysts microinjected with 1 MASC (Table5). The degree of chimerism ranged between 0.1% to >45%. After 6 to 20weeks, animals were sacrificed. Some mice were frozen in liquid nitrogenand thin sections were cut as described. Whole-mouse sections werestained with X-Gal. One thousand sets of digital images coveringcompletely each section were then assembled to create a composite imageof each whole-mouse section. In a representative non-chimeric animal (byQ-PCR for NEO) derived from a blastocyst in which a single MASC wasinjected, no X-Gal staining was seen. In contrast, the animal was 45%chimeric by R-PCR for NEO by tail clip analysis and had contribution ofa single ROSA26-derived MASC to all somatic tissues.

For other animals, multiple organs were harvested and analyzed for thepresence of MASC derived cells by X-GAL staining, staining with ananti-β-gal-FITC antibody, and Q-PCR for NEO. Animals that had NEO⁺ cellsin tail-clippings had contribution of the ROSA26-derived MASC in alltissues, including the brain, retina, lung, cardiac and skeletal muscle,liver, intestine, kidney, spleen, BM, blood, and skin as shown by X-GALstaining and staining with an anti-β-gal-FITC antibody.

Chimerism was detected by X-Gal staining and anti-β-gal staining in theanimals generated from blastocysts microinjected with ROSA26 MASC.β-gal⁺ cells expressed markers typical for the tissue in which they hadincorporated. β-gal⁺ cells co-stained with anti-β-gal⁺ FITC andanti-NF200 or GFAP and TOPRO3 (observed at 20× magnification) for NF200and GFAP in the central nervous system and for dystrophin in theskeletal muscle. Lung tissue was stained for anti-β-gal-FITC and pan-CKin alveoli and bronchi (also TOPRO3) (observed at 20× magnification).Skeletal muscle was stained with anti-β-gal-FITC, dystrophin-PE, andTOPRO3 was observed at 20× magnification. Heart was stained withanti-β-gal-FITC and cardiac troponin-I-Cy3, TOPRO3 was observed at 20×magnification. Liver was stained with anti-β-gal-FITC and pan-CK-PE andTOPRO3 (was observed with 40× magnification and 10× magnification).Intestine was stained with anti-β-gal-FITC, pan-CK-PE, and TOPR03 wasobserved at 20× magnification. Kidney was stained with anti-β-gal-FITC(glomerulus, tubulus) was observed at 20× magnification. Marrow stainingwas observed with anti-β-gal-FITC and CD45-PE, GR1-PE and MAC1-PE.Spleen staining was observed with anti-β-gal-FITC and CD45-PE, CD3-PEand CD 19-PE. Levels of engraftment estimated by Q-PCR for NEO Wereconcordant with those estimated by X-GAL and anti-β-gal-FITC staining.

Summary

These data demonstrate for the first time that BM derived single MASCintegrate into the developing mouse, giving rise to cells of variousfates, and contributing to the generation of all tissues and organs ofthe three germ layers of the mouse. As all live animals, irrespective ofthe degree of chimerism, had normal functioning organs, these studiesalso suggest that MASC can differentiate in vivo in functional cells ofthe three germ layers. Whether MASC contribute to germ cells, wheninjected in a blastocyst or when injected postnatally, has not yet beentested.

Example 9 Origin of Endothelial Progenitors

Vasculogenesis, the in situ differentiation of primitive endothelialprogenitors, termed angioblasts, into endothelial cells that aggregateinto a primary capillary plexus is responsible for the development ofthe vascular system during embryogenesis (Hirashima et al., 1999). Incontrast, angiogenesis, defined as the formation of new blood vessels bya process of sprouting from preexisting vessels, occurs both duringdevelopment and in postnatal life (Holash et al., 1999; Yang et al.,2001). Until recently, it was thought that blood vessel formation inpost-natal life was mediated by sprouting of endothelial cells fromexisting vessels. However, recent studies have suggested thatendothelial “stem cells” may persist into adult life, where theycontribute to the formation of new blood vessels (Peichev et al., 2000;Lin et al., 2000; Gehling et al., 2000; Asahara et al., 1997; Shi etal., 1998), suggesting that like during development neoangiogenesis inthe adult may at least in part depend on a process of vasculogenesis.Precursors for endothelial cells have been isolated from BM andperipheral blood (Peichev et al., 2000; Watt et al., 1995). The ontogenyof these endothelial progenitors is unknown.

During development, endothelial cells are derived from mesoderm. TheVEGF receptor 2, Flk1, characterizes the hemangioblasts, a bipotent stemcell found in the aorto-gonad-mesonephros region (Medvinsky et al.,1996; Fong et al., 1999; Peault, 1996) and in fetal liver (Fong et al.,1999), and commitment of embryoid bodies to hemangioblasts isaccompanied with expression of Flk1 (Choi et al., 1998; Choi, 1998).Whether hemangioblasts persist in adult life is not known, and only HSCand endothelial progenitors have, been documented. Like hemangioblasts,endothelial progenitors express Flk1 (Peichev et al., 2000) and onereport suggested that HSC in post-natal life express Flk1 (Ziegler etal., 1999). During embryology, commitment of the hemangioblast to theendothelial lineage is characterized by the sequential expression ofVE-cadherin, CD31, and shortly afterwards CD34 (Nishikawa et al., 1998;Yamashita et al., 2000). In post-natal life, endothelial progenitorshave been selected from BM and blood using Abs against AC133, Flk1,CD34, and the H1P12 Ab (Peichev et al., 2000; Lin et al., 2000; Gehlinget al., 2000). AC133 has also been found on other cells, including NSCs(Uchida et al., 2000) and gastrointestinal epithelial cells (Corbeil etal., 2000). Upon differentiation to mature endothelium, the AC133receptor is quickly lost (Peichev et al., 2000; Gehling et al., 2000).Another receptor found on circulating endothelial cells is a mucin,MUC18, recognized by the H1P12 Ab (Lin et al., 2000). MUC18 is lost upondifferentiation of endothelial progenitors to endothelium. CD34 isexpressed on endothelial progenitors, as well as on hematopoieticprogenitors (Peichev et al., 2000; Baumhueter et al., 1994) and hepaticoval cells (Crosby et al., 2001). This antigen is also lost upondifferentiation of endothelial progenitors to endothelium. Most matureendothelial cells, but microvascular endothelial cells, no longerexpress CD34.

It is described here for the first time, the in vitro generation of vastnumbers of endothelial cells that engraft in vivo and contribute toneoangiogenesis from a MASC. MASC can be culture expanded for >80 PDsand endothelial cells generated from MASC can be expanded for at leastand additional 20 PDs. MASC may therefore be an ideal source ofendothelial cells for clinical therapies. In addition, as MASC areontogenically less mature than the “angioblast”, this model should beuseful to characterize endothelial commitment and differentiation.

hMASC Differentiate into Cells with Phenotypic Characteristics ofEndothelium

MASC were obtained and cultured as described in Example 3. To induceendothelial differentiation, MASC were replated at 2×10⁴ cells/cm² inFN-coated wells in serum-free expansion medium without EGF and PDGF-BBbut with 10 ng/mL VEGF. In some instances, FCS was added. Cultures weremaintained by media exchange every 4-5 days. In some instances, cellswere subcultured after day 9 at a 1:4 dilution under the same cultureconditions for 20+ PDs.

In order to define endothelial differentiation from MASC moreextensively, FACS and immunohistochemical analysis of cells after 3-18days was performed. Expression of Flk1 and Flt1 on undifferentiated MASCwas low, was maximal at day 9, and persisted until day 18. VE-cadherin,present on BM or blood endothelial progenitors (Peichev et al., 2000;Nishikawa et al., 1998), was not expressed on undifferentiated MASC, butwas expressed after 3 days of culture with VEGF and persisted until day18. MASC expressed low levels of AC133, found on endothelial as well ashematopoietic progenitors (Peichev et al., 2000; Gehling et al., 2000)but was no longer detectable after day 3. CD34, present on endothelialand hematopoietic progenitors (Peichev et al., 2000; Asahara et al.,1997; Rafii et al., 1994), was not present on undifferentiated MASC(FIG. 4A) but was expressed from day 9 until day 18. The mucin, MUC18,recognized by the Ab H1P12 has been used to purify endothelialprogenitors from blood (Lin et al., 2000). Although MASC did not stainwith H1P12 MASC treated with VEGF for 9 days stained positive, butexpression was lost by day 18.

The endothelium specific integrin, αvβ3, (Eliceiri et al., 2000) was notpresent on undifferentiated MASC, whereas αvβ5 was expressed at very lowlevels. Expression of integrins increased progressively duringdifferentiation and was maximal by day 14 (FIG. 5). The tyrosine kinasereceptors, Tie and Tek, important for angiogenesis but not endothelialcell differentiation (Partanen et al., 1999), were not expressed onMASC. Expression of Tek could be seen after day 3 and Tie after day 7(FIG. 6). MASC also do not express vWF, but vWF was expressed from day 9on (Rosenberg et al., 1998; Wagner et al., 1982). More matureendothelial markers, including CD31, CD36, CD62-P (Tedder et al., 1995)(FIG. 7), and the adhesion junction proteins ZO-1, β-catenin, andγ-catenin (FIG. 5) were detected after day 14 (Li et al., 1990; VanRijen et al., 1997; Petzelbauer et al., 2000). VCAM or CD62-E were notexpressed at high level at any time point during differentiation, unlessendothelium was activated with IL-1α, as described below.Differentiation to endothelium was associated with acquisition ofβ2-microglobulin and low levels of HLA-class I antigens, but notHLA-class II.

It has been reported previously, that endothelial differentiation canonly be obtained from MASC expanded with 2% FCS or less, but not whenexpanded with 10% FCS (Reyes et al., 2001) as higher concentrations ofFCS supports growth of classical MSC that differentiate only intoosteoblasts, chondroblasts and adipocytes (Reyes et al., 2001; Pittengeret al., 1999). When FCS was present during the initial 7 days ofdifferentiation, endothelial differentiation could not be induced. Whennon-confluent MASC (≤1×10⁴ cells/cm²) were induced to differentiate,endothelial was not seen. When MASC were subcultured 9-days afterexposure to VEGF using serum free medium with 10 ng/mL VEGF, cells couldundergo at least an additional 12 PDs. When 10% FCS and 10 ng/mL VEGFwas added to the medium for subculturing, MASC-derived endothelial cellscould undergo an additional 20+ PDs, irrespective of the number of PDsthat MASC had undergone.

Compared with undifferentiated MASC, endothelial cells were larger, andhad a lower nuclear/cytoplasm ratio. Results were similar when MASC wereused from cultures that had undergone 20 (n=30) or 50+ (n=25) PDs.

Functional Characteristics of MASC-Derived Endothelium

It was tested whether VEGF-induced differentiated progeny of hMASC hadfunctional characteristics of endothelial cells. Endothelial cellsrespond to hypoxia by upregulating expression of VEGF as well as theVEGF receptors Flk1 and the angiogenesis receptors, Tie-1 and Tek(Kourembanas et al., 1998). hMASC and hMASC-derived endothelial cellswere incubated at 37° C. in 20% or 10% O₂ for 24 h. Cells were stainedwith Abs against Flk1, Flt1, Tek and IgG control, fixed in 2%paraformaldehyde and analyzed by flow cytometry. In addition, VEGFconcentrations in the culture supernatants was measured using an ELISAkit (AP biotech, Piscataway, N.J.). MASC-derived endothelial cells andundifferentiated MASC were exposed to hypoxic conditions for 24 h.

Expression of Flk1 and Tek was significantly increased on MASC-derivedendothelial cells exposed to hypoxia (FIG. 7), while the levels of thesereceptors remained unchanged on undifferentiated MASC. In addition,levels of VEGF in culture supernatants of hypoxic endothelial cultureswas increased by 4 fold (FIG. 7B) whereas VEGF levels in MASC culturesexposed to hypoxia remained unchanged.

It was next tested whether MASC-derived endothelial cells wouldupregulate expression of HLA-antigens and cell adhesion ligands inresponse to inflammatory cytokines, such as IL-1α (Meager, 1999; Steeberet al., 2001). 10⁶ MASC and MASC-derived endothelial cells wereincubated with 75 ng/ml IL-1α (R&D Systems) in serum-free medium for 24h. Cells were fixed in 2% paraformaldehyde and stained with Abs againstHLA-Class I, class II, β2-microglobulin, vWF, CD31, VCAM, CD62E andCD62P, or control Abs, and analyzed using a FACScalibur (BectonDickinson).

Significantly increased levels of HLA-Class I and II, β2-microglobulin,VCAM, ECAM, CD62E, CD62P were seen by FACS analysis (FIG. 7C) onendothelial cells. In contrast, on undifferentiated MASC onlyupregulation of Flk was seen.

Another characteristic of endothelial cells is that they take up LDL(Steinberg et al., 1985). This was tested by incubating MASC induced todifferentiate with VEGF for 2, 3, 5, 7, 9, 12 and 15 and 21 withLDL-dil-acil. The dil-Ac-LDL staining kit was purchased from BiomedicalTechnologies (Stoughton, Mass.). The assay was performed as permanufacture's recommendations. Cells were co-labeled either withanti-Tek, -Tie-1 or -vWF Abs. After 3 days, expression of Tek wasdetected but no uptake of a-LDL. After 7 days, cells expressed Tie-1,but did not take up significant amounts of a-LDL. However, acquisitionof expression of vWF on day 9 was associated with uptake of aLDL (FIG.6B).

Endothelial cells contain vWF stored in Weibel Pallade bodies that isreleased in vivo when endothelium is activated (Wagner et al., 1982).This can be induced in vitro by stimulating cells with histamine(Rosenberg et al., 1998), which also results in activation of the cellcytoskeleton (Vischer et al., 2000). MASC-derived endothelial cells wereplated at high confluency (10⁴ cells/cm²) in FN-coated chamber slides.After 24 h, cells were treated with 10 μM histamine (Sigma) in serumfree medium for 25 min. and stained with Abs against vWF and myosin.Untreated and treated cells were fixed with methanol at −20° C. for 2min, stained with Abs against vWF and myosin, and analyzed usingfluorescence and/or confocal microscopy. vWF was present throughout thecytoplasm of untreated endothelial cells. Cytoplasm of endothelial cellstreated with histamine contained significantly less vWF and vWF was onlydetectable in the perinuclear region, likely representing vWF present inthe endoplasmic reticulum (FIG. 6A). Histamine treatment caused wideningof gap junctions and cytoskeletal changes with increased numbers ofmyosin stress fibers (FIG. 6A).

Finally, endothelial cells were tested to determine if they could form“vascular tubes” when plated on Matrigel™ or extracellular matrix (ECM)(Haralabopoulos et al., 1997). 0.5 ml extracellular matrix (Sigma) wasadded per well of a 24 well plate, incubated for 3 h at 37° C. 10⁴ MASCand MASC-derived endothelial cells were added per well in 0.5 ml ofserum free plus VEGF medium and incubated at 37° C. As shown in FIG. 6C,culture of MASC derived endothelial cells on ECM resulted in vasculartube formation within 6 hours.

hMASC-Derived Endothelial Cells Contribute to Tumor-Angiogenesis In Vivo

A breeding colony of NOD-SCID mice was established from mice obtainedfrom the Jackson Laboratories (Bar Harbor, Me.). Mice were kept inspecific pathogen free conditions and maintained on acidified water andautoclaved food. Trimethoprim 60 mg and sulphamethoxazole 300 mg(Hoffmann-La Roche Inc., Nutley, N.J.) per 100 ml water was given twiceweekly.

Three Lewis lung carcinoma spheroids were implanted subcutaneously inthe shoulder. 3 and 5 days after implantation of the tumor, mice wereinjected with 0.25×10⁶ human MASC-derived endothelial cells or humanforeskin fibroblasts via tail vein injection. After 14 days, animalswere sacrificed, tumors removed and cryopreserved using OTC compound(Santura Finetek USA Inc, Torrance, Calif.) at −80° C. In addition, theears that were clipped to tag the mouse were also removed andcryopreserved using OTC compound at −80° C. Five μm thick sections ofthe tissues were mounted on glass slides and were fixed and stained asdescribed below.

Computer-aided analysis of length and number of branches counted on fivesections of each tumor showed that tumors in mice that received humanMASC-derived endothelial cells had a 1.45±0.04 fold greater vascularmass than tumors in control mice that did withanti-human-β2-microglobulin or HLA-Class I Abs, combined withanti-mouse-anti-CD31 Abs and anti-vWF, anti-Tek or anti-Tie-1 Abs, whichrecognize both human and mouse endothelial cells. These initial studiesshowed that some blood vessels in the tumor containedanti-human-β2-microglobulin or HLA-Class I positive cells thatco-labeled for either vWF, Tie or Tek, but not with mouse-CD31,indicating that human MASC-derived endothelial cells contributed totumor neoangiogenesis in vivo.

To better address the degree of contribution, 35 sequential 5 μm slideswere obtained and were stained in an alternate fashion with eitheranti-human β2-microglobulin-FITC or anti-mouse-CD31-Cy5 andanti-vWF-Cy3. All slides were examined by confocal microscopy. Thedifferent figures were then assembled in 3-D to determine the relativecontribution of human and murine endothelial cells to the tumor vessels.When tumors obtained from animals injected with human-MASC derivedendothelial cells were analyzed approximately 35% of the tumor vesselswere positive for anti-human β2-microglobulin as well as vWF whereasapproximately 40% of endothelial cells stained positive with anti-mouseCD31 Abs (FIG. 8A-G). Tumors in animals that did not receive endothelialcells or received human fibroblasts did not contain endothelial cellsthat stained positive with the anti-β2-microglobulin or anti-HLA-class-IAbs Abs.

MASC-derived endothelial cells were also analyzed whether theycontribute to wound healing angiogenesis. The area of the ear that hadbeen clipped to tag the mouse was then examined. Neoangiogenesis in theear wounds was in part derived from the MASC derived endothelial cells.Similar to blood vessels in the tumor the percent human endothelialcells present in the healed skin wound was 30-45% (FIG. 9H).

Undifferentiated hMASC Differentiate in Endothelial Cells In Vivo

10⁶ undifferentiated MASC were injected I.V. in 6-week old NOD-SCIDmice. Animals were maintained for 12 weeks and then sacrificed. In oneanimal, a thymic tumor was detected, which is commonly seen in agingNOD-SCID mice (Prochazka et al., 1992). The thymus was removed andcryopreserved in OTC compound at −80° C. Ten μm thick sections of thetissues were mounted on glass slides and were fixed and stained asdescribed below.

All hematopoietic cells stained positive for mouse CD45 but not humanCD45, indicating that they were murine in origin. The tumor was thenstained with an anti-human β2-microglobulin-FITC Ab and an anti-vWF-Cy3Ab that recognizes both human and mouse endothelial cells. Approximately12% of the vasculature was derived from hMASC (FIG. 9I). These studiesfurther confirmed that the hematopoietic elements were not of humanorigin, as no human β2-microglobulin was detected outside of thevascular structures.

Immunohistochemistry and Data Analysis

In vitro cultures: Undifferentiated MASC or MASC induced todifferentiate to endothelium for 2-18 days, plated in FN coated chamberslides were fixed with 2% paraformaldehyde (Sigma) for 4 min at roomtemperature. For cytoskeleton staining chamber slides were fixed withmethanol for 2 min at −20° C. For intracellular ligands, cells werepermeabilized with 0.1 Triton-X (Sigma) for 10 min and incubatedsequentially for 30 h in each with primary antibody (Ab), and FITC, PEor Cy5 coupled anti-mouse-, goat- or rabbit-IgG Ab. Between each step,slides were washed with PBS+1% BSA. Primary Abs against CD31, CD34,CD36, CD44, HLA-class I and -II, β2-microglobulin were used at a 1:50dilution. Primary Abs against VCAM, ICAM, VE-cadherin, selectins, HIP12,ZO-1, connexin-40, connexin-43, MUC18, a_(v)b₃, a_(v)b₅, B-catenin andγ-catenin (Chemicon) and Tek, Tie, vWF (Santa Cruz) were used at a 1:50dilution. Stress fibers were stained with Abs against myosin (lightchain 20 kD, clone no. MY-21; 1:200). Secondary Abs were purchased fromSigma and used at the following dilutions: anti-goat IgG-Cy-3 (1:40),anti-goat IgG-FITC (1:160), anti-mouse IgG-Cy-3 (1:150) and anti-mouseIgG-FITC (1:320), anti-rabbit-FITC (1:160) and anti-rabbit-Cy-3 (1:30).TOPRO-3 was purchased from Sigma. Cells were examined by fluorescencemicroscopy using a Zeiss Axiovert scope (Carl Zeiss, Inc., Thomwood,N.Y.) as well as by confocal fluorescence microscopy using a Confocal1024 microscope (Olympus AX70, Olympus Optical Co. LTD, Japan).

Tumors or normal tissue: The tissue was sliced using a cryostat in 5-10μm thick slices. Slices were fixed with acetone for 10 min at roomtemperature and permeabilized with 0.1 Triton X for 5 min. Slides wereincubated overnight for vWF, Tie or Tek, followed by secondaryincubation with FITC, PE or Cy5 coupled anti-mouse-, goat- or rabbit-IgGAbs and sequential incubation with Abs against mouse CD45-PE or humanCD45-FITC, human β2-microglobulin-FITC, mouse CD31-FITC or TOPRO-3 for60 min. Between each step, slides were washed with PBS+1% BSA. Slideswere examined by fluorescence microscopy using a Zeiss Axiovert scope aswell as by confocal fluorescence microscopy using a Confocal 1024microscope. 3D-reconstruction consisted of the collection of sequential0.5 μm confocal photos from 35 slides of 5 μm thick to a total of 350photos. Slides were stained with vWF-Cy3 and alternately double stainedwith humanβ2-microglobulin-FITC or mouse CD31-FITC. The photos from eachslide were aligned with the next slide in Metamorph software (UniversalImaging Corp) and the 3D reconstruction was made in 3D Doctor Software(Able software Corp).

Volume of relative contribution of human (green) and murine endothelialcells (false colored as blue) to the 3D vessel was calculated as cubicpixels of each color. The area of each color was also calculated assquare pixels in 4 vessels through the 35 slides to obtain an accuratepercentage of contribution. The area of each color was also calculatedin alternate slides of four different tumors.

Summary

The central finding of this study is that cells that co-purify with MSCfrom BM have the ability to differentiate to endothelial cells that havein vitro functional characteristics indistinguishable from matureendothelial cells. It is also showy that such endothelial cellscontribute to neoangiogenesis in vivo in the setting of wound healingand tumorigenesis, and that undifferentiated MASC can respond to localcues in vivo to differentiate into endothelial cells contributing totumor angiogenesis. As the same cell that differentiates to endotheliumalso differentiates to other mesodermal cell types, as well as cells ofnon-mesodermal origin, the cell defined here precedes the angioblast,and even the hemangioblast in ontogeny.

It has also been shown that MASC differentiate into cells that expressmarkers of endothelial cells, but proved that VEGF induced MASC functionlike endothelial cells. Endothelial cells modify lipoproteins duringtransport in the artery wall (Adams et al., 2000). Endothelial cellsmaintain a permeability barrier through intercellular junctions thatwiden when exposed to hemodynamic forces or vasoactive agents, such ashistamine (Rosenberg et al., 1998; Li et al., 1990; Van Rijen et al.,1997; Vischer et al., 2000). Endothelial cells release prothromboticmolecules such as vWF, tissue factor, and plasminogen activatorinhibitor to prevent bleeding (Vischer et al., 2000), and regulateegress of leukocytes by changing expression levels of adhesion moleculesin response to inflammation (Meager, 1999; Steeber et al., 2001).Endothelium also reacts to hypoxia by producing VEGF and expressing VEGFreceptor aimed at increasing vascular density (Kourembanas et al.,1998). Therefore it has been demonstrated that endothelial cellsgenerated from MASC can perform all of these tasks when tested in vitro.

Finally it has been proved that in vitro generated MASC-derivedendothelial cells respond to angiogenic stimuli by migrating to thetumor site and contributing to tumor vascularization as well as woundhealing in vivo. This finding confirms that endothelial cells generatedfrom MASC have all the functional characteristics of mature endothelium.The degree of contribution of endothelial cells to tumor angiogenesisand neo-angiogenesis was 35-45%, levels similar to what has beendescribed for other sources of endothelial cells (Conway et al., 2001;Ribatti et al., 2001). In addition, it has been found that angiogenicstimuli in vivo provided in a tumor microenvironment are sufficient torecruit MASC to the tumor bed and induce their differentiation intoendothelial Cells that contribute to the tumor vasculature. Thesestudies therefore extend studies reported by other groups demonstratingthat cells present in marrow can contribute to new blood vesselformation (Peichev et al., 2000; Lin et al., 2000; Gehling et al., 2000;Asahara et al., 1997), in a process similar to vasculogenesis, precursorresponsible for this process has been identified the. This is to ourknowledge the first report that identifies a cell present in post-natalBM as a very early progenitor for endothelial cells.

Example 10 Derivation of Neurons

Single adult BM-derived hMASC or mMASC were tested to determine whetherthey can differentiate ex vivo to functional neurons, as well astrocytesand oligodendrocytes aside from mesodermal cell types. mMASC and hMASCwere selected and culture expanded as previously described in Examples 1and 3, respectively. Human neural progenitor cells (hNPC) were purchasedfrom Clonetics (San Diego, Calif.). hNPC were cultured anddifferentiated per manufactures' recommendations.

Electrophysiology: Standard whole-cell patch-clamp recording was used toexamine the physiological properties of MASC-derived neurons.Voltage-clamp and current-clamp recordings were obtained using a Dagan3900A patch-clamp amplifier (Dagan Corporation, Minneapolis) which wasretrofitted with a Dagan 3911 expander unit. Patch pipettes, made fromborosilicate glass, were pulled on a Narishige pipette puller (modelPP-83), and polished using a Narishige microforge (model MF-83). Patchpipettes were filled with an intracellular saline consisting of (in mM)142.0 KF, 7.0 Na₂S0₄, 3.0 MgSO₄, 1.0 CaCl₂, 5.0 HEPES, 11.0 EGTA, 1.0glutathione, 2.0 glucose, 1.0 ATP (magnesium salt), 0.5 GTP (sodiumsalt). For most recordings, the fluorescent dye 5,6-carboxyfluorescein(0.5 mm; Eastman Kodak Chemicals) was also added to the pipette solutionto confirm visually, using fluorescence microscopy, that the whole-cellpatch recording configuration had been achieved. Pipette resistancesranged from 11 to 24 Mohm. The standard extracellular recording salinewas comprised of the following (in mM): 155 NaCl, 5.0 KCl, CaCl₂, 1.0MgCl₂, 10 HEPES, 5 glucose. For some experiments 1 μM TTX was added tothe standard control solution. The pH of the intracellular andextracellular recording solutions was adjusted to 7.4 and 7.8,respectively, using NaOH. All chemicals were from Sigma. PClamp 8.0(Axon Instruments, Foster City) was used to run experiments, and tocollect and store data. The data presented were corrected for a 8.4 mVliquid junctional potential, which was calculated using the JPCALCsoftware package. Off-line analyses and graphical representations of thedata were constructed using Clampfit 8.0 (Axon Instruments, Foster City)and Prism (Graphpad, San Diego).

Transduction: Retroviral supernatant was produced by incubatingMFG-eGFP-containing PG13 cells, provided by Dr. G. Wagemaker, U. ofRotterdam, Netherlands (Bierhuizen et al., 1997), with MASC expansionmedium for 48 h, filtered and frozen at −80° C. MASC were incubated withretroviral supernatants and 8 μg/ml protamine (Sigma) for 6 h. This wasrepeated 24 h later. Transduction efficiency was analyzed by FACS.

Gene microarray analysis: RNA was isolated from hMASC, bFGF orFGF-8b+EGF induced cells using the RNeasy mini kit (Qiagene), digestedwith DNase I (Promega) at 37° C. for 1 h and re-purified using theRNeasy. The [³²P] dATP labeled cDNA probe, generated according to themanufacturers recommendations, was hybridzed to the Human NeurobiologyAtlas Array (Clonetech #7736-1, Clonetech Laboratories, Palo Alto,Calif., USA) at 68° C. for 18-20 h, followed by 4 washes in 2×SSC, 1%SDS at 68° C. for 30 min each time, 0.1×SSC, 0.5% SDS at 68° C. for 30min, and once in 2×SSC at room temperature for 5 min. The arrays wereread by a phosphorimager screen scanner (Molecular Dynamics, Storm 860)and analyzed using Atlas Image 1.0 (Clontech). Differences betweenundifferentiated and differentiated cells greater than 2-fold wereconsidered significant.

PCR analysis for retroviral insert: PCR was used o amplify the flankingsequence 3′ from the 3′ LTR of the MFG vector in the human genomic DNA.DNA from 10⁶ MASC or endothelial, myoblast or neuroectodermaldifferentiated progeny was prepared from cells by standard methods. 300ng of genomic DNA was digested with AscI and a splinkerette linker wasligated to the 5′ end (Devon R. S. et al., 1995). The twooligonucleotides used for the splinkerette linker were as follows:aattTAGCGGCCGCTTGAATTtttttttgcaaaaa, (the hairpin loop forming sequenceis in lower case and the upper case is the reverse complement of thesecond splinkerette oligo), andagtgtgagtcacagtagtctcgcgttcgAATTAAGCGGCCGCTA, (the underlined sequenceis also the sequence of the linker-specific primer (LS Primer) used forthe PCR and RT steps). A 5′-biotin-T7 coupled primer was used thatrecognizes a sequence in the eGFP gene[Biotin-ggc-cag-tga-att-gta-ata-cga-ctc-act-ata-ggc-tgg-CAC-ATG-GTC-CTG-CTG-GAG-TTC-GTG-AC;underlined portion shows the minimum promoter sequence needed forefficient in vitro transcription and the upper case is the eGFP specificsequence] and LS primer to amplify the flanking regions for 10 roundsusing Advantage 2 polymerase (Clontech). The biotin labeled amplifiedproduct was captured using streptavidin-magnetic beads (StreptavidinMagnetic Particles; Roche) and the resultant product was furtheramplified using the T7 RNA polymerase an approximately 1,000 fold andthen DNAase 1 treated. The resultant product was reverse transcribedusing the agtgtgagtcacagtagtctcgcgttc splinkerette primer according tothe superscript II protocol (Gibco), and subsequently amplified by 30rounds of nested PCR using the primer for the 3′LTR [ggc caa gaa cag atggaa cag ctg aat atg]. The flanking sequence in the human genome fromendothelium, muscle, and neuroectodermal differentiated cells andundifferentiated MASC was sequenced.

To demonstrate that the same insertion site was present in multipledifferentiated progeny, specific primers were generated in thehost-flanking genome. Real time PCR amplification (ABI PRISM 7700,Perkin Elmer/Applied Biosystems) was used to quantitate the flankingsequence compared to the eGFP sequence. Reaction conditions foramplification were as follows: 40 cycles of a two step PCR (95° C. for15 sec, 60° C. for 60 sec) after initial denaturation (95° C. for 10min.) with 2 μl of DNA solution, 1× TaqMan SYBR GreenUniversal Mix(Perkin Elmer/Applied Biosystems) PCR reaction buffer. Primers used wereas follows: Clone A16: LTR primer=CCA-ATA-AAC-CCT-CTT-GCA-GTT-G;Flanking sequence chromosome 7=TCC-TGC-CAC-CAG-AAA-TAA-CC; Clone A 12chromosome 7 sequence: LTR primer=GGA-GGG-TCT-CCT-CTG-AGT-GAT-T,Flanking sequence=CAG-TGG-CCA-GAT-CTC-ATC-TCA-C; Clone A12 chromosome 1sequence: LTR=GGA-GGG-TCT-CCT-CTG-AGT-GAT-T; Flankingsequence=GCA-GAC-GAG-GTA-GGC-ACT-TG. The relative amount of the flankingsequence was calculated in comparison with eGFP sequence according tomanufacturer's recommendations using the 7700 ABI PRISM DetectorSoftware 1.6.

Neural transplantation: Newborn (P1-P3) male Sprague Dawley rats(Charles River Laboratories) were used in this study. The rats wereanaesthetized by cryoanesthesia. The cranium was immobilized using amodified stereotaxic head holder and the scalp reflected to expose theskull. hMASC were harvested with 0.25% trypsin/EDTA, washed twice, andresuspended in PBS. The viability of the hMASC was more than 85%. A 2 μlvolume of hMASC suspended in phosphate buffered saline at aconcentration of 0.7×10⁴ cells/μl was stereotaxically injectedintracerebroventricularly with a tapered glass micropipette attached toa Hamilton syringe using the following coordinates (mm from bregma): AP−0.6, ML 0.8, DV 2.1, toothbar was set at −1. Following the injections,the scalp was sutured and the pups allowed to recover.

Four and 12 weeks after transplantation, the rats were anaesthetizedwith chloral hydrate (350 mg/kg, i.p.), decapitated the brains removed,frozen in powered dry ice, and stored at −80° C. Fresh frozen brainswere sectioned using a cryostat and fixed with 4% paraformaldehyde for20 min immediately before staining. Sections were incubated for one hourat room temperature with blocking/permeabilization solution consistingof 2% normal donkey serum (Jackson Immuno Labs) and 0.3% triton X.Primary and secondary antibodies were diluted in the sameblocking/permeabilization solution for subsequent steps. Primaryantibodies (mouse anti human nuclei (1:25), anti human nuclear membrane(1:25) and anti NeuN (1:200) from Chemicon; rabbit anti GFAP (1:250)from DAKO, rabbit anti NF200 (1:300) from Sigma were incubated overnightat 4° C., rinsed 3×10 minutes each in PBS and followed by secondary Cy3(1:200) anti and FITC (1:100) antibodies (all from Jackson Immuno Labs)for two hours at room temperature. Slides were examined by fluorescencemicroscopy using a Zeiss Axiovert scope as well as by confocalfluorescence microscopy using a Confocal 1024 microscope.

hMASC Acquire a Neuron, Astrocyte and Oligodendrocyte Phenotype whenCultured with bFGF.

Neuroectodermal differentiation was done as described in Example 5.Briefly, cells were cultured in FN-coated chamberslides or cultureplates with serum-free medium consisting of 60% DMEM-LG, 40% MCDB-201(Sigma Chemical Co, St Louis, Mo.), supplemented with 1×ITS, 1×LA-BSA,10⁻⁸ M dexamethasone, 10⁻⁴M ascorbic acid 2-phosphate (AA) (all fromSigma), 100 U penicillin and 1,000 U streptomycin (Gibco). In somecultures, we added 100 ng/mL bFGF whereas in other cultures 10 ng/mLEGF+10 ng/mL FGF-8b were added (all from R&D Systems). Cells were notsubcultured, but media was exchanged every 3-5 days.

Two weeks after re-plating with bFGF, 26±4% of cells expressed astrocyte(GFAP+), 28±3% oligodendrocyte (MBP+) and 46±5% neuron (NF200+) markersas shown in Table 6.

TABLE 6 Differentiation markers on bFGF and FGF-8b induced hMSC bFGFbFGF bFGF FGF-8b FGF-8b FGF-8b (day 7) (day 14) (day 21) (day 7) (day14) (day 21) GFAP 36 ± 4% 26 ± 4% 0 0 0 0 MBP 35 ± 3% 28 ± 3%  4 ± 2% 00 0 GaIC 30 ± x% 26 ± 5%  8 ± 3% 0 0 0 Nestin 35 ± 6%  6 ± 3% Not tested 90 ± 10% 10 ± 6% Not tested Neuro-D 20 ± 2% 0% Not tested 50 ± 6% Nottested Not tested Tuji 30 ± 3% 23 ± 5% 23 ± 2% 88 ± 5% 92 ± 3% 98 ± 2%PSA- 33 ± 2% 16 ± 3% Not tested 40 ± 7% Not tested Not tested NCAM NF680 26 ± 7% 22 ± 3% 0 20 ± 3% Not tested NF160 0 46 ± 5% 50 ± 3% 0 65 ± 3%Not tested NF200 0 15 ± 2% 22 ± 5% 0 75 ± 8% 92 ± 6% NSE 0 40 ± 4% 82 ±5% 0 78 ± 3% 80 ± 8% MAP2-AB 0 40 ± 6% 80 ± 2% 0 95 ± 4% 95 ± 3% Tau 028 ± 2% 78 ± 7% 0 93 ± 2% 92 ± 4% GABA 0 0 0 0 39 ± 4% 40 ± 2%Parvalbumin 0 0 0 0 28 ± 6% 35 ± 3% TH 0 0 0 20 ± 5% 23 ± 5% 25 ± 6% DCC0 0 0 0 25 ± 6% 28 ± 2% DTP 0 0 0 0 35 ± 7% 38 ± 3% TrH 0 0 0 0 26 ± 6%25 ± 4% Serotonin 0 0 0 0 30 ± 5% 35 ± 3% Nurrl 0 0 0 0 20 ± 4% 23 ± 2%c-ret 0 0 0 0 33 ± 3% 35 ± 5%

When hMASC were replated at higher cell densities (2×10⁴ cells/cm²) toinduce differentiation, no cells with neuroectodermal phenotype could bedetected, suggesting that cell-cell interactions prevent bFGF-inducedneuroectodermal differentiation.

The distribution of astrocyte-, oligodendrocyte- and neuron-like cellsdid not differ when differentiation was induced with hMASC that hadundergone 20 or 60 PDs. However, when hMASC expanded for 20 PDs werecultured with bFGF, >50% of cells died while >70% of hMASC cultureexpanded for >30 PDs survived and acquired a neuron-, astrocyte- oroligodendrocyte-like phenotype. This suggests that not all hMASC can beinduced to acquire neural characteristics but that a subpopulation ofhMASC that survives long-term in vitro may be responsible for neuronaldifferentiation. It has been shown that the karyotype of hMASC is normalirrespective of culture duration (Reyes et al., 2001). Differentiationof hMASC into neuroectodermal-like cells is therefore not likely due totransformation of MASC following long-term culture.

Most astrocyte- and oligodendrocyte-like cells died after 3 weeks.Progressive maturation of neuron-like cells was seen throughout culture.After 1 week, bFGF treated hMASC stained positive for NeuroD, Nestin,polysialated neural cell adhesion molecule (PSA-NCAM), and tubulin-β-III(TuJI) (Table 6). After 2 weeks, bFGF treated cells stained positive forNF68, -160, and -200, NSE, MAP2-AB, and Tau. bFGF-induced neurons didnot express markers of GABA-ergic, serotonergic or dopaminergic neurons,but expressed glutamate as well as the glutamate-receptors-5, -6 and -7and N-methyl-D-aspartate (NMDA)-receptor, and Na⁺-gated voltagechannels.

Further confirmation of neuroectodermal differentiation was obtainedfrom cDNA array analysis of two independent hMASC populations induced todifferentiate for 14 days with 100 ng/mL bFGF. Expression levels ofnestin, otx 1 and otx2Consistent with the immunohistochemicalcharacterization, a >2 fold increase in mRNA for nestin was detected,GFAP, glutamate-receptors 4, 5, and 6, and glutamate, and severalsodium-gated voltage channels, but did not detect increases in TH or TrHmRNA levels. A >2 fold increase in mRNA levels was also found formammalian achaete-scute homolog 1 (MASH I) mRNA, a transcription factorfound only in brain (Franco Del Arno et al., 1993) and ephrin-A5 mRNA(O'Leary and Wilkinson, 1999). The astrocyte specific markers GFAP andS100A5, and oligodendrocyte specific markers, myelin-oligodendrocyteglycoprotein precursor and myelin protein zero (PMZ), as well asHuntingtin, and major prion protein precursor mRNA wereexpressed >2-fold higher after exposure to bFGF. A greater than 2 foldincrease was also seen for several glycine receptors, GABA-receptors,the hydroxytryptophan receptor-A and neuronal acetylcholine receptor,glycine transporter proteins, synaptobrevin and synaptosomal-associatedprotein (SNAP)25. Finally, bFGF induced expression of BDNF andglia-derived neurotrophic factor (GDNF).

Like hMASC, mMASC Acquire a Neuron, Astrocyte and OligodendrocytePhenotype when Cultured with bFGF.

MASC derived from other species was tested to determine whether similarresults could be obtained. mMASC expanded for 40-90 PDs were replated at10⁴ cells/cm² in conditions identical to those used for hMASC. After 14days, mMASC acquired morphologic and phenotypic characteristics ofastrocytes (GFAP⁺), oligodendrocytes (MBP⁺) and neurons (NF-200⁺, NSE⁺and Tau). NF200 and GFAP or MBP were never found in the same cell. Incontrast to undifferentiated mMASC, mMASC treated with bFGF weresignificantly larger and extended processes for >40 μm.

To determine whether neuron-like cells had functional characteristics ofneurons, and if bFGF-induced cells showed evidence of voltage-gated Na⁺currents a patch clamp was used. No sodium currents or fast spikingbehavior was seen in any of the mMASC derived neuron-like cells (n=59),even though some cells expressed calcium currents, and in 4 cells therewas evidence of spiking behavior mediated by calcium currents. Thus,bFGF induced cells did not have functional voltage-gated Na⁺ currents,despite expression of sodium-gated voltage channel mRNA and protein.

hMASC Acquire a Midbrain Dopaminergic, Serotonergic and GABAergicPhenotype when Cultured with EGF and FGF-8b.

FGF-8b, expressed at the mid-hindbrain boundary and by the rostralforebrain, induces differentiation of dopaminergic neurons in midbrainand forebrain and serotonergic neurons in the hindbrain (Ye et al.,1998). In vitro, FGF-8b has been used to induce dopaminergic andserotonergic neurons from murine ES cells (Lee et al., 2000).

hMASC (n=8), expanded=for 20 to 60 PDs, were replated at 2×10⁴ cells/cm²on FN in serum free medium with ITS and AA and with 10 ng/mL FGF-8b and10 ng/mL EGF. More than 80% of cells survived for 3 weeks. FGF-8b andEGF induced differentiation into cells staining positive for neuronalmarkers (Table 6) (day 7: PSA-NCAM, Nestin and TuJ1; day 14: NF-68,NF-160, NF-200; and day 21: MAP2-AB, NSE, Tau, and Na⁺-gated voltagechannels) but not oligodendrocytes and astrocytes. In contrast to ourobservation for bFGF induced differentiation, cells plated at 10⁴cells/cm² with EGF and FGF-8b did not lead to differentiation. After 2-3weeks, cells had characteristics of GABAergic (GABA⁺, parvalbumin⁺),dopaminergic (TH⁺, DCC⁺, and DTP⁺) and serotonergic (TrH⁺ andserotonin⁺) neurons (Table 6). Cells also expressed the GABA-A-receptorand glutamate receptors. Cells with a dopaminergic phenotype alsostained positive with Abs against the nuclear transcription factor,Nurr1, expressed only in midbrain dopaminergic neurons (Saucedo-Cardenaset al., 1998) as well as the proto-oncogene cRet, a membrane-associatedreceptor protein tyrosine kinase, which is a component of the glial cellline-derived neurotrophic factor (GDNF) receptor complex expressed ondopaminergic neurons (Trupp et al., 1996). This suggests that FGF-8binduces a phenotype consistent with midbrain dopaminergic neurons.

Again, results from immunohistochemical studies were confirmed by cDNAarray analysis on hMASC induced to differentiate for 14 days withFGF-8b+EGF. Consistent with the immunohistochemical characterization,a >2 fold increase in mRNA for TH, TrH, glutamate, severalglutamate-receptors, and sodium-gated voltage channels was detected. Asparvalbumin and GABA are not present on the array, their expressioncould not be confirmed by mRNA analysis. Consistent with the almostexclusive neural differentiation seen by immunhistochemnistry, there wasno increase in expression of GFAP, S100A5 mRNA nor mRNA for theoligodendrocyte specific marker, PMZ. FGF-8b+EGF induced cellsexpressed >2 fold more tyrosine kinase receptor (Trk)A, BDNF and GDNF,several glycine-, GABA- and hydroxytryptamine-receptors, and severalsynaptic proteins.

Coculture with the Glioblastoma Cell Line U87 Enhances NeuronMaturation.

Irrespective of the culture conditions used, hMASC-derived neurons didnot survive more than 3-4 weeks in culture. As neither culture containedglial cells after 3 weeks, it is possible that neuronal cells thatexpress both glutamate and glutamate-receptors died due to glutamatetoxicity (Anderson and Swanson, 2000). Alternatively, factors requiredfor neural cell survival, differentiation and maturation provided byglial cells might not be present in the cultures (Blondel et al., 2000;Daadi and Weiss, 1999; Wagner et al., 1999). To test this hypothesis,cells from 3-week old FGF-8b+EGF cultures were cocultured with theglioblastoma cell line, U-87, in serum-free medium supplemented withFGF-8b+EGF for an additional 2 weeks.

The glioma cell line, U-87, [American Tissue Cell Collection (Rockville,Md.)] was maintained in DMEM+10% FCS (Hyclone Laboratories, Logan,Utah). Cells from 3-week old FGF-8b+EGF containing cultures were labeledwith the lipophylic dye, PKH26 (Sigma), as per manufacturer'srecommendations. Labeled cells were replated in FN coated chamber slidesin FGF-8b+EGF containing serum free medium together with 1,000 U-87cells and maintained an additional 2-3 weeks with media changes every3-5 days. To assure that PKH26 present in MASC-derived cells did nottransfer to the U-87 cell line, U-87 cells were cultured inBSA-containing medium and 20 μl PKH26 dye for 7 days. No labeling ofglioma cells was detected.

Under these serum-free conditions, U-87 cells ceased to proliferate butsurvived. hMASC derived neurons were labeled with the membrane dye,PKH26, prior to coculture with U-87 cells to allow us to identify thehMASC-derived cells by fluorescence microscopy. FGF-8b+EGF inducedneurons cocultured after 3 weeks with U-87 cells and the same cytokinessurvived for at least 2 additional weeks. Neurons acquired a more maturemorphology with increased cell size as well increased number, length andcomplexity of the neurites.

The electrophysiological characteristics of PKH26 labeled neural cellsderived from hMASC after coculture with U-87 cells by whole-cell currentclamp and voltage-clamp after current-injection was evaluated (FIG. 9B).Current-clamp demonstrated spiking behavior in response to injectedcurrent in 4/8 of PKH26 labeled hMASC-derived cells present inFGF-8b+EGF/U-87 cultures. The resting membrane potential of spiking andnon-spiking cells was −64.9±5.5 mV and −29.7±12.4 mV, respectively. Foreach cell studied, input resistance of spiking and non-spiking cells was194.3 (37.3) and 216.3 (52.5) Mohm, respectively. An example of one ofthe cells in which w observed spiking behavior is shown in FIG. 9B. Thetop panel shows a family of voltage traces which was elicited from aspiking cell under control conditions. A DC current was first injectedin the cell to hold them in the range of −100 to −120 mV. A currentinjection protocol, as shown in the middle panel, was then used to drivethe membrane potential to different levels. As shown in this example,depolarizing current steps that were sufficiently large to bring thecell to threshold for action potential, evoked a single spike. The lowerpanel shows that the spiking behavior of the cells could be blocked by 1μM TTX, suggesting that the action potentials are mediated by Na-gatedvoltage channels. Leak-subtracted current records, obtained involtage-clamp mode from the same cells (FIG. 9C), demonstrated an inwardcurrent that was transient in time course and activated at voltages morepositive than −58 mV, as well as outward currents. The transient inwardcurrent was blocked reversibly by 1 μM TTX. This pharmacology, togetherwith the transient time course and the voltage-dependent activation ofthe inward current is typical for voltage-gated Na⁺ currents, found onlyin mature neurons and skeletal muscle cells (Sah et al., 1997;Whittemore et al., 1999). Skeletal muscle markers in these neuron-likecells was not detected. These studies suggest that treatment withFGF-8b+EGF and co-culture with glioblastoma cellsfresults ininaturationto cells with the fundamental characteristics of excitable neurons,TTX-sensitive voltage-gated Na⁺ currents.

hMASC Transplanted in Ventricles of Newborn Rats Differentiate in CellsExpressing Astrocyte and Neuronal Markers

1.4×10⁴ hMASC were stereotactically injected in the lateral ventriclesof P1-P3 Sprague Dawley rats. After 4 and 12 weeks, animals weresacrificed and analyzed for presence of human cells and evidence ofdifferentiation of hMASC to neuroectoderm. Human cells, identified bystaining with a antibodies against human nuclei or human nuclearmembrane could be seen in the SVZ up to 400 μm away from the ventriclein animals analyzed after 4 weeks, and after 12 weeks, human cells couldalso be seen deeper in the brain parenchyma such as in the hippocampusand along the fornix. Some human cells had typical astrocyte morphologyand stained positive with anti-GFAP antibodies, whereas other cellsstained positive with anti-Neu-N antibodies, NF-200 and anti-humannuclear membrane antibodies. Triple staining showed that human nuclearantigen positive Neu-N positive cells did not coexpress and GFAP.

Summary

The central finding of this work is that single post-natal BM-derivedMASC can be induced to differentiate not only into mesodermal cell typesbut also cells with mature neuronal characteristics, as well asastrocyte and oligodendrocyte characteristics. Time-dependent as well asculture-method-dependent maturation of MASC to cells withneuroectodermal features was shown. Double staining definitivelydemonstrated that neuronal or glial cells were authentic and resultswere not due to inappropriately expressed neuronal or glial markers.These results were confirmed at the mRNA level. Retroviral markingstudies were used to demonstrate that the neurons, astrocytes andoligodendrocytes were derived from a single MASC that alsodifferentiates into muscle and endothelium, as the sequence of the hostcell genomic region flanking the retroviral vector was identical in alllineages. hMASC did not only acquire phenotypic but alsoelectrophysiological characteristics of mature neurons, namelyTTX-sensitive voltage-gated Na⁺ currents. Finally, it was also shownthat MASC can differentiate in vivo into cells expressing neuronal andastrocyte markers.

Using retroviral marking of hMASC combined with PCR-based sequencing ofthe genomic sequence flanking the 3′-LTR of the retroviral insert, itwas shown that neurons are derived from the same hMASC thatdifferentiate into astrocytes and oligodendrocytes, as well as intoendothelium and muscle (Jordan et al., 1990). This conclusivelydemonstrates that MASC can, at the single cell level, differentiate tocells of mesodermal and neuroectodermal lineages. The cells with theability to differentiate not only into mesodermal cell types but alsoneuroectodermal cell types multipotent adult stem cells, or MASC werere-named. Sanchez-Ramos et al. (Sanchez-Ramos et al., 2000) and Woodburyet al. (Woodbury et al., 2000) showed that populations of human orrodent MSC can express markers of astrocytes and neurons, but notoligodendrocytes in vitro. However, neither study provided evidence thatthe same cell that acquired neuroectodermal markers could alsodifferentiate into mesodermal cells. Furthermore, neither study showedthat cells expressing neuronal markers also acquired functional neuronalcharacteristics. Thus, although suggestive for neural differentiation,these reports did not conclusively demonstrate neural and glialdifferentiation from MSC.

It was also shown that hMASC transplanted in the ventricle of newbornrats can migrate in the neurogenic subventricular zone and into thehippocampus where they respond to local cues to differentiate into cellsexpressing astrocyte and neuronal markers. This model was chosen becausemigration and differentiation of NSC to specific neuronal phenotypesoccurs to a much greater extent when transplantation is done in germinalareas of the brain than in non-neurogenic areas, and when transplantsare done in newborn animals compared with adult animals (Bjorklund andLindvall, 2000; Svendsen and Caldwell, 2000). Although hMASC aremultipotent and differentiate into cells outside of the neuroectoderm,hMASC did not form teratomas. The number of cells that had migratedoutside the subventricular area was low after 4 weeks, but increasedafter 12 weeks.

The ease with which MASC can be isolated from post-natal BM, expandedand induced to differentiate in vitro to astrocytes, oligodendrocytes orneuronal cell types may circumvent one of the key problems in NSCtransplantation, namely the availability of suitable donor tissue.

Example 11 MASC Differentiation into Hepatocyte-Like Cells

During embryogenesis, the first sign of liver morphogenesis is athickening of the ventral endodermal epithelium, which occurs betweene7.5 and e8.5 in the mouse (Zaret K. S., 2001). Little is known aboutthe signals involved in initial endoderm formation and subsequentendoderm specification. Early in gastrulation (e6-e7) endoderm is notspecified, not even in an anterior/posterior direction (Melton D.,1997). However, recent studies showed that ex vivo exposure of endodermto FGF4 posteriorizes the early endoderm, which is now competent toexpress hepatic markers (Wells J. M. et al., 1999). By e8.5 in themouse, definitive endoderm has formed the gut tube and expresses HNF3β(Zaret K. S., 2000). The foregut endoderm is induced to the hepatocytelineage by acidic (a) FGF and bFGF, both produced by the adjacentcardiac mesoderm (Zaret K. S., 2001), which are required to induce ahepatic fate and not the default pancreatic fate (Zaret K. S., 2001).Basic morphogenetic proteins (BMP's) produced by the transversummesenchyme are also required as they increase levels of the GATA4transcription factor which promote the ability of endoderm to respond toFGF's (Zaret K. S., 2001). GATA4 and HNF3β are required for hepaticspecification and are important effectors of downstream events leadingto hepatocyte differentiation, as they upregulate markers of hepatocytespecific expression such as albumin, among others.

In most instances, mature hepatocytes can undergo several cell divisionsand are responsible for hepatic cell replacement. As a result, there hasbeen great controversy about the existence and function of a liver stemcell. During extensive liver necrosis due to chemical injury or whenhepatocytes are treated with chemicals that block their proliferation, apopulation of smaller cells with oval shape, termed oval cells, emergesand proliferates (Petersen, B. E., 2001). These oval cells mayconstitute the “stem cell” compartment in the liver. Oval cells residein the smallest units of the biliary tree, called the canals of Herring,from where they migrate into the liver parenchyma (Theise N. D., et al.,1999). Oval cells are bi-potential, giving rise in vitro and in vivo toboth hepatocytes and bile ductular epithelium. Oval cells expressseveral hematopoietic markers such as Thy1.1, CD34, Flt3-receptor, andc-Kit, and also express αFP, CK19, γ-glutamyl-transferase, and OV-6. Theorigin of oval cells is not known (Petersen, B. E., 2001; Kim T. H. etal, 1997; Petersen, B. E., 2001).

Until recently, it was believed that hepatocytes could only be derivedfrom cells of endodermal origin and their progenitors. However, recentstudies suggest that non-endodermal cells may also form hepatocytes invivo and in vitro (Petersen, B. E., 2001; Pittenger M. F. et al., 1999).Following bone marrow (BM) transplantation, oval cells are derived fromthe donor BM (Theise N. D., et al., 1999). Transplantation of enrichedhematopoietic stem cells (HSC) in FAH^(−/−) mice, an animal model oftyrosenimia type I, resulted in the proliferation of large numbers ofdonor LacZ⁺ hepatocytes and animals had restored biochemical function ofthe liver (Lagasse E. et al., 2000). Furthermore, single HSC may notonly repopulate the hematopoietic system but also contribute toepithelium of lung, skin, liver and gut (Krause D. S. et al., 2001).Exocrine pancreatic tumor cells treated in vitro with dexamethasone(Dex) with or without oncostatin M (OSM) may acquire a hepatocytephenotype (Shen C. N. et al., 2000). Finally, mouse embryonic stem (ES)cells spontaneously acquire a hepatocyte phenotype, a process that isenhanced by treatment with aFGF, HGF, OSM, and Dex (Hamazaki T. et al.,2001).

It was demonstrated here that single MASC not only differentiate intomesodermal and neuroectodermal cells, but also into cells withmorphological, phenotypic and functional characteristics of hepatocytesin vitro.

mMASC, rMASC, and hMASC Acquire a Hepatocyte-Like Phenotype whenCultured with FGF4 and/or HGF.

mMASC, rMASC and hMASC were selected and cultured as described. Todetermine optimal conditions for MASC differentiation intohepatocyte-like cells, the effect of different extracellular matrix(ECM) components was tested and cytokines known to induce hepatocytedifferentiation in vivo or from ES cells (Zaret K. S., 2001) on mMASC orrMASC differentiation to hepatocytes. As differentiation requires cellcycle arrest, the effect of cell density was also tested. To demonstratedifferentiation to hepatocyte like cells, cells were stained after 14days with Abs against albumin, CK18, and HNF3β.

Optimal differentiation of mMASC or rMASC to albumin, CK18 and HNF3βpositive epithelioid cells was seen when MASC were plated at 21.5×10³cells/cm² in the presence of 10 ng/ml FGF4 and 20 ng/ml HGF on Matrigel™as shown in Table 7A. After 14 days, the percent albumin, CK18 and HNF3βpositive epithelioid cells was 61.4±4.7%, and 17.3% of cells werebinucleated. When plated on FN, differentiation to CK18 and HNF3βpositive epithelioid cells was also seen, even though only 53.1±6.3% ofcells stained and fewer (10.9%) binucleated cells were seen.

Culture with either FGF4 or HGF yielded albumin, CK18 and HNF3β positiveepithelioid cells, but the percent albumin, CK18 and HNF3β positivecells was higher when mMASC or rMASC were treated with both FGF4 and HGFas shown in Table 7A. Addition of aFGF, bFGF, FGF7, BMP's, or OSM didnot increase the percent cells positive for hepatocyte markers, while 1%DMSO and 0.1 mM-10 mM Sodium Butyrate did not support differentiation ofmMASC or rMASC to cells positive for hepatocyte markers.

When cell densities between 2.5 and 25×10³ cells/cm² were tested, thehighest percent cells with hepatocyte markers was seen in culturesseeded at 21.5×10³ cells/cm². No hepatocyte differentiation was seenwhen cells were plated at <12.5×10³ cells/cm².

hMASC were plated at 3-30×10³ cells/cm² on 10 ng/mL FN or 1% Matrigel™with aFGF, bFGF, FGF7, 1% DMSO, HGF, and/or FGF4. Only cells treatedwith 10 ng/ml FGF4 alone, 20 ng/ml HGF alone, or a combination of bothdifferentiated into epithelioid cells that expressed albumin, CK18 andHNF3β. hMASC plated at 15-30×10³ cell/cm² differentiated intoepithelioid cells whereas hMASC plated at 3×10³ cell/cm² died. LikemMASC or rMASC, the percent albumin, CK18 and HNF3β positive epithelioidcells was higher when hMASC were cultured on Matrigel™ (91.3%±4.4) thanon FN (89.5%±5.4), and the percent binucleated cells was higher onMatrigel™ (31.3%) than on FN (28.7%) as shown in Table 7B.

TABLE 7 Optimization of MASC differentiation into hepatocyte like cells.A: Mouse and Rat B: Human FGF-4 HGF FGF4 + HGF FGF-4 HGF FGF-4 + HGF FNAlbumin ++/++ ++/+ ++/++ +++++ +++++ +++++ CK18 ++/++ ++/+ +++/++  ++++++++++ +++++ HNF3β +++/+++ +++/+  ++++/+++  +++++ NT +++++ Matrigel ™Albumin ++/++  +/+ +++/+++ +++++ NT +++++ CK18 ++/++ ++/+ +++/+++ +++++NT +++++ HNF3β +++/+++ +++/++ ++++/++++ +++++ NT +++++ Collagen Albumin− − − NT NT NT CK18 − − − NT NT NT HNF3β − − − NT NT NT − = 0% + = 20%,++ = 30%, +++ = 40%, ++++ = 60%, +++++ = 80% cells staining positive forspecific markers and NT = not tested.Phenotypic Characterization of MASC Differentiation to Hepatocyte-LikeCells

Hepatocyte differentiation was further evaluated over time byimmunofluorescence and confocal microscopy for early (HNF3β, GATA4,CK19, αFP) and late (CK18, albumin, HNF1α) markers of hepatocytedifferentiation. mMASC or rMASC plated on Matrigel™ with FGF4 and HGFenlarged from 8 μm to 15 μm diameter as shown in Table 8A. On d21-d28,approximately 60% of cells were epithelioid and surrounded by smallerround or spindle shaped cells. Undifferentiated mMASC or rMASC did notexpress any of the liver specific transcription factors or cytoplasmicmarkers. After 4 days, cells expressed HNF3β, GATA4 and αFP, low levelsof CK19, and very rare cells stained positive for HNF1α, albumin orCK18. At seven days, the large epithelioid cells stained positive forHNF3β, GATA4, HNF1α with increasing staining for albumin and CK18. Onlyrare cells expressed αFP. After 14, 21 and 28 days, the largeepithelioid cells stained positive for GATA4, HNF3β, HNF1α, CK18 andalbumin, but not αFP or CK19. The smaller cells surrounding the nodulesof epithelioid cells stained positive for CK19 and αFP.

hMASC was plated on Matrigel™ with FGF4 and HGF or FGF4 alone enlargedfrom 10-12 μm to 20-30 μm diameter by d21. After 7 days, cells expressedHNF3β, GATA4 and low levels of CK19, while few cells stained positivefor albumin or CK18. After 14 and 21 days, >90% of epithelioid cellsstained positive for GATA4, HNF3β, HNF1α, HNF4, CK18 and albumin, whileonly rare cells stained positive for αFP or CK19 as shown in FIG. 10B.

TABLE 8 Immunohistochemistry Pattern of Hepatocyte Marker Expression A:Mouse and Rat B: Human D4 D7 D10 D14 D21 D4 D7 D10 D14 D21 HNF3β +/+ +/++/+ +/+ +/+ NT + NT + + Gata4 +/+ +/+ +/+ +/+ +/+ NT + NT + + α-FP +/++/+ NT/NT −/− −/− NT + NT − − HNF1α −/− +/+ NT/NT +/+ +/+ NT − NT + +Albumin −/− +/+ +/+ +/+ +/+ NT + NT + + CK18 −/− +/+ +/+ +/+ +/+ NT −NT + + + = Marker is expressed, − = Marker is not expressed and NT = nottestedHepatocyte-Like Cells are Derived from the Same Single hMASC thatDifferentiated into Neuroectoderm and Endoderm

It has been shown that single mMASC or rMASC differentiate intoendothelium, neuroectoderm and CK18 and albumin positive endodermalcells. It has also been shown that single hMASC differentiate intomesoderm and neuroectoderm. The same single hMASC was tested todetermine whether they can differentiate into hepatocyte-like cells.MASC were obtained, cultured and expanded as described. Fordifferentiation, mMASC or rMASC were plated at 5-25×10³ cells/cm² on0.01-10 μg/ml fibronectin (FN), 0.01-8 μg/ml collagen (Sigma ChemicalCo, St. Louis, Mo.), or 1% Matrigel™ (Becton-Dickinson) in serum-freemedium [60% low glucose DMEM (DMEM-LG; Gibco-BRL, Grand Island, N.Y.),40% MCDB-201 (Sigma), supplemented with 1× insulin/transferrin/selenium,4.7 μg/ml linoleic acid, 1 mg/ml bovine serum albumin (BSA), 10⁻⁸ Mdexamethasone, 10⁻⁴ M ascorbic acid 2-phosphate (all from Sigma), 100U/ml penicillin, 100/ml U streptomycin (Gibco)], with 2% FCS (Hyclone,Logan Utah) and 10 ng/mL each epidermal growth factor (EGF) (Sigma),leukemia inhibitory factor (LIF; Chemicon, Temecula, Calif.), andplatelet derived growth factor (PDGF-BB; R&D Systems, Minneapolis,Minn.). hMASC were plated at 15-30×10³ cells/cm² on 0.1 μg/ml FN, or 1%Matrigel™ in the same medium without LIF (Reyes M., 2002). After 8-12 h,media were removed, cells washed twice with phosphate buffered saline(PBS) (Fischer) and cultured in serum-free medium supplemented with 5-50ng/ml HGF, aFGF, bFGF, FGF4, FGF7, or OSM; or 10 mg/mldimethyl-sulphoxide (DMSO), or 0.1-1 mM sodium butyrate.

Transduction of hMASC with eGFP was performed using an eGFP-cDNAcontaining retrovirus and expanded to >5×10⁶ cells. Twenty percent wasinduced to differentiate into muscle, endothelium, neuroectoderm andendoderm. For clone A16 a single retroviral insertion site was presentin undifferentiated MASC as well as mesodermal and neuroectodermaldifferentiated cells and eGFP⁺ clone A16 MASC differentiated into CK18and albumin positive cells. The same insertion site was present inFGF4-treated MASC generated from the same cell population (5′-TAGCGGCCGCTTGAATTCGAACGCGAGACTACTGTGACT CACACT-3′, Chromosome 7), provingthat single hMASC differentiate into endoderm aside from mesoderm andneuroectoderm.

Quantitative RT-PCR Demonstrates that FGF4 and HGF Induces HepatocyteSpecification and Differentiation.

Hepatocyte differentiation by quantitative RT-PCR was confirmed forearly (HNF3β, GATA4, CK19, αFP) and late (CK18, albumin, HNF1α,cytochrome P450) markers of hepatocyte differentiation. RNA wasextracted from 3×10⁵ MASC or MASC induced to differentiate tohepatocytes. mRNA was reverse transcribed and cDNA was amplified asfollows: 40 cycles of a two step PCR (95° C. for 15″, 60° C. for 60″)after initial denaturation (95° C. for 10′) with 2 μl of DNA solution,1× TaqMan SYBR Green Universal Mix PCR reaction buffer using a ABI PRISM7700 (Perkin Elmer/Applied Biosystems). Primers used for amplificationare listed in Table 9.

TABLE 9 Primers used Primer Name Primers- MOUSE HNFαS: 5′-TTCTAAGCTGAGCCAGCTGCAGACG-3′ A: 5′-GCTGAGGTTCTCCGGCTCTTTCAGA-3′HNF3β S: 5′-CCAACATAGGATCAGATG-3′ A: 5′-ACTGGAGCAGCTACTACG-3′ GATA4S: 5′-AGGCATTACATACAGGCTCACC-3′ A: 5′-CTGTGGCCTCTATCACAAGATG-3′ CK18S: 5′-TGGTACTCTCCTCAATCTGCTG-3′ A: 5′-CTCTGGATTGACTGTGGAAGTG-3′ CK19S: 5′-CATGGTTCTTCTTCAGGTAGGC-3′ A: 5′-GCTGCAGATGACTTCAGAACC-3′ AlbuminS: 5′-TCAACTGTCAGAGCAGAGAAGC-3′ A: 5′-AGACTGCCTTGTGTGGAAGACT-3′ αFPS: 5′-GTGAAACAGACTTCCTGGTCCT-3′ A: 5′-GCCCTACAGACCATGAAACAAG-3′ TTRS: 5′-TCTCTCAATTCTGGGGGTTG-3′ A: 5′-TTTCACAGCCAACGACTCTG-3′ Cyp2b9S: 5′-GATGATGTTGGCTGTGATGC-3′ A: 5′-CTGGCCACCATGAAAGAGTT-3′ Cyp2b13S: 5′-CTGCATCAGTGTATGGCATTTT-3′ A: 5′-TTTGCTGGAACTGAGACTACCA-3′ HUMANαFP S: 5′-TGCAGCCAAAGTGAAGAGGGAAGA-3′ A: 5′-CATAGCGAGCAGCCCAAAGAAGAA-3′Albumin   S: 5′-TGCTTGAATGTGCTGATGACAGGG-3′A: 5′-AAGGCAAGTCAGCAGGCATCTCATC-3′ CK19 S: 5′-ATGGCCGAGCAGAACCGGAA-3′A: 5′-CCATGAGCCGCTGGTACTCC-3′ CK18 S: 5′-TGGTACTCTCCTCAATCTGCTG-3′A: 5′-CTCTGGATTGACTGTGGAAGT-3′ CYP1B1 S: 5′-GAGAACGTACCGGCCACTATCACT-3′A: 5′-GTTAGGCCACTTCAGTGGGTCATGAT-3′ CYP2B6 S: 5′-GATCACACCATATCCCCGGA-3′A: 5′-CACCCTACCACCCATGACCG-3′ RAT HNF1α S: 5′-AGCTGCTCCTCCATCATCAGA-3′A: 5′-TGTTCCAAGCATTAAGTTTTCTATTCTAA-3′ HNF3β S: 5′-CCTACTCGTACATCTCGCTCATCA-3′ A: 5′-CGCTCAGCGTCAGCATCTT-3′ CK18S: 5′-GCCCTGGACTCCAGCAACT-3′ A: 5′-ACTTTGCCATCCACGACCTT-3′ CK19S: 5′-ACCATGCAGAACCTGAACGAT-3′ A: 5′-CACCTCCAGCTCGCCATTAG-3′ Albumin S: 5′-CTGGGAGTGTGCAGATATCAGAGT-3′ A: 5′-GAGAAGGTCACCAAGTGCTGTAGT-3′ αFPS: 5′-GTCCTTTCTTCCTCCTGGAGAT-3 ′ A: 5′-CTGTCACTGCTGATTTCTCTGG-3′ TTRS: 5′-CAGCAGTGGTGCTGTAGGAGTA-3′ A: 5′-GGGTAGAACTGGACACCAAATC-3′ CYP2b1S: 5′-GAGTTCTTCTCTGGGTTCCTG-3′ A: 5′-ACTGTGGGTCATGGAGAGCTG-3′

mRNA levels were normalized using β-actin (mouse and human) or 18S (rat)as housekeeping genes and compared with mRNA levels in freshly isolatedrat or mouse hepatocytes, rat hepatocytes cultured for 7 days, or mRNAfrom human adult liver RNA purchased from Clontech, Palo Alto, Calif.

On d0, mMASC and rMASC expressed low levels of albumin αFP, CK18, CK19,TTR, HNF3β, HNF1α and GATA4 mRNA, but no CYP2B9 and CYP2B13 (mouse) orCYP2B1 (rat) mRNA (FIG. 10). Following treatment of mMASC or rMASC withFGF4 and HGF, expression of HNF3β and GATA4 mRNA increased on d2, becamemaximal by d4, decreasing slightly and leveling off by d14. mRNA forαFP, and CK19 increased after d2, and became maximal by d4 and decreasedthereafter. TTR mRNA increased after d4, was maximal by d7 and leveledoff. CK18, Albumin, HNF1α and P450 enzyme mRNA increased after d7 andwas maximal on d14. Between d14 and d21, FGF4 and HGF induced MASCexpressed albumin, TTR, CK18, CYP2B9 and CYP2B13 (mouse) and CYP2B1(rat) mRNA.

Undifferentiated hMASC expressed low levels of albumin, CK18, and CK19,CYP1B1, but not αFP (FIG. 10) and CYP2B6 mRNA. Levels of albumin, CK18,CK19, CYP1B1 mRNA increased significantly in hMASC cultured with FGF4alone or with FGF4 and HGF for 14 days compared to day 0 (MASC)cultures. Levels of albumin, CK18 and CYP1B1 mRNA continued to increaseand were higher on d28. Although, CYP1B1 is not a specific hepatocytemarker, its upregulation suggests hepatocyte commitment and maturation.Low levels of CYP2B6, 0.5% to 1.0% of fresh liver mRNA's could be seenon d14 and d21 but not d0. mRNA levels of immature hepatocyte markers(CK19 and αFP) decreased as differentiation progressed and were higherin cultures induced with FGF4 alone, whereas mRNA levels for maturehepatocytes (CK18 and albumin) were higher in FGF4 and HGF-inducedhMASC.

Western Blot Demonstrates that FGF4+HGF Induces Hepatocyte Specificationand Differentiation

Expression of hepatocyte-specific genes was also confirmed by WesternBlot and performed as described by Reyes et al. (2000). Abs to αFP,human albumin, CK18 were diluted 1:1000 in blocking buffer. Goatanti-β-actin (1:1000) was from Santa Cruz. Secondary Abs were HRP-linkedgoat anti-mouse and HRP-linked donkey anti-goat (Amersham, ArlingtonHeights). ECL was performed according to manufacturers instructions(Amersham). Undifferentiated hMASC did not express CK18, albumin, or αFPprotein (FIG. 10B). After treatment for 35 days with FGF4 alone or FGF4and HGF, hMASC expressed albumin and CK18, but not aFP, consistent withthe histochemical analysis.

mMASC, rMASC and hMASC Acquire Hepatocyte Functional Activity

Five different assays were used to determine whether cells withmorphologic and phenotypic characteristics of hepatocytes also hadfunctional hepatocyte attributes.

Urea production and secretion by hepatocyte-like cells was measured atvarious time points throughout differentiation. Urea concentrations weredetermined by colorimetric assay (Sigma Cat. 640-1) per manufacturer'sinstructions. Rat hepatocytes grown in monolayer and fetal mouse liverbuds were used as positive controls, and culture medium as negativecontrol. The assay can detect urea concentrations as low as 10 mg/ml. Asthe assay also measures ammonia, samples were assessed before and afterurease addition.

No urea or ammonia was detected in culture medium alone.Undifferentiated MASC did not produce urea. Following treatment withFGF4 and HGF, urea production by MASC increased in a time dependentmanner. The time course for urea production in mouse and rat cultureswas similar. For hMASC treated with FGF4 and HGF, urea was not detectedon d4, was similar to mouse and rat cultures by d12, and exceeded thatin mouse or rat cultures on d21. Levels of urea produced by MASC-derivedhepatocytes were similar to that in monolayer cultures of primary rathepatocytes. For all three species, significantly more urea was producedby cells differentiated on Matrigel™ compared to FN.

Albumin production was measured at various time points throughout thedifferentiation. Rat albumin concentrations were determined by acompetitive enzyme linked immunoassay (ELISA) described previously(Tzanakakis E. S., et al., 2001; Wells J. M. et al., 2000). Human andmouse albumin concentrations were determined using a similar ELISAmethod with substitution of human or mouse albumin and anti-human oranti-mouse albumin Abs for the rat components where appropriate.Peroxidase conjugated anti-human-albumin and reference human albuminwere from Cappel. Peroxidase conjugated and affinity purified anti-mousealbumin and reference mouse albumin were from Bethyl Laboratories(Montgomery, Tex.). To ensure specificity of the ELISA, human, mouse,and rat Abs were incubated for 2 hrs at 37° C. with 3% BSA in distilledwater (dH₂O). ELISA's had a sensitivity of at least 1 ng/ml.

Undifferentiated MASC did not secrete albumin. Following treatment withFGF4 and HGF, mMASC, rMASC and hMASC produced albumin in a timedependent manner. As was seen for urea production, MASC differentiatedon Matrigel™ produced higher amounts of albumin than when cultured onFN. Mouse, rat, and human cells secreted similar levels of albumin, eventhough albumin was not detected in human cultures on d3. Levels ofalbumin produced by mouse, rat and human MASC-derived hepatocytes weresimilar to those seen in monolayer cultures of primary rat hepatocytes.

Cytochrome P450 activity was next assessed in aggregates of MASC-derivedhepatocytes and primary rat liver hepatocyte spheroids using the PRODassay. mMASC-hepatocyte aggregates were formed by plating d14 FGF4 andHGF treated mMASC at 5×10⁴ cells/cm² on non-tissue culture plates, whichwere placed on a shaker at 10 revolutions per minute for 5 h. Cellaggregates were transferred to Primaria™ dishes and allowed to compactfor 4 days in the presence or absence of 1 mM phenobarbital.hMASC-hepatocyte aggregates were formed by hanging drop method. Briefly,10³ hMASC treated for 24 days with FGF4 and HGF were placed into 100 μLdrops with or without 1 mM phenobarbital. After 4 days, aggregates werecollected and cytochrome P450 activity assessed by PROD assay.Pentoxyresorufin (PROD) (Molecular Probes, Eugene, Oreg.) isO-dealkylated by Cytochrome P450, changing a non-fluorescent compoundinto a fluorescent compound, resorufin (Tzanakakis E. S. et al., 2001).Fluorescence intensity caused by PROD metabolism consequently estimatescytochrome P450 (CYP) activity. Assessment and detection of resorufin insitu was performed using confocal microscopy as described (Tzanakakis E.S. et al., 2001).

No PROD activity was seen in aggregates of undifferentiated mMASC orhMASC. However, mMASC (18 days with FGF4 and HGF) and hMASC (28 days,FGF4 alone) induced to form aggregates had significant PROD activity.PROD activity in MASC-derived hepatocyte aggregates was similar to thatof primary rat hepatocyte aggregates. A number of different cells haveP450 activity, but P450 activity up-regulation by phenobarbital is onlyseen in hepatocytes. Therefore, P450 was also tested in the presence orabsence of phenobarbital. Without phenobarbital, several P450 enzymespartially participate in PROD metabolism giving an inflated fluorescencevalue for those samples. In contrast, in the phenobarbital inducedaggregates, PROD activity is almost wholly metabolized by mousecytochromes Cyp2b9, Cyp2b10, and Cyp2b13, rat cytochrome Cyp2b1/2(Tzanakakis E. S. et al., 2001), and in human, by CYP2B6. Thereforeincreased fluorescent activity is smaller than the actual increase inthe protein expression of the stated cytochrome P450 enzymes. Whenaggregates were cultured for 96 hours with phenobarbital, a 32% to 39%increase in PROD activity was seen, suggesting presence of functionalhepatocyte specific Cyp2b9, Cyp2b10, and Cyp2b13 in mMASC and CYP2B6 inhMASC-derived hepatocytes.

MASC-derived hepatocytes were also assessed for their ability to take upLDL by incubating FGF4 treated hMASC with LDL-dil-acil. Cells wereco-labeled either with anti-CK18 or anti-Pan-CK and HNF-3β or GATA4 Abs.After 7 days, low level uptake of a-LDL was detected, which increased tobecome maximal on d21.

Another metabolic function of hepatocytes is glycogen production orgluconeogenesis. The levels of glycogen storage were analyzed byperiodic acid Schiff (PAS) staining of FGF4 and HGF induced mouse MASCand FGF induced hMASC at d3, d7, d14, and d21. For PAS, slides wereoxidized in 1% periodic acid for 5′ and rinsed 3 times in dH₂O.Afterwards slides were treated with Schiff's reagent for 15′, rinsed indH₂O for 5-10′, stained with Mayer's hematoxylin for 1′ and rinsed indH₂O. Glycogen storage was first seen by d14 and maximum levels wereseen after d21 (FIG. 11).

Hepatocyte Isolation and Culture

Hepatocytes were harvested from 4-6 week old male Sprague-Dawley rats asdescribed (Seglen P.O., 1976). Hepatocyte viability after the harvestranged from 90-95%. Hepatocytes were cultured as described (TzanakakisE. S. et al., 2001; Tzanakakis E. S. et al., 2001). To form a monolayer,hepatocytes were cultured on 35 mM Fischer culture plates (FischerScientific, Pittsburgh, Pa.) coated with 8 μg/cm² collagen (CohesionTechnologies, Palo Alto, Calif.). To form spheroids, hepatocytes werecultured on 35-mm Primaria™ dishes (Becton Dickinson). Under bothconditions, seeding density was 5×10⁴ cells/cm². 12 h after initialplating, medium was changed to remove dead and unattached cells. Mediumwas replaced every 48 hours thereafter.

Summary

It has been shown that a single post-natal mouse, rat and humanBM-derived MASC can differentiate in vitro into an endodermal cell typewith hepatocyte phenotype and function. MASC, cultured under hepatocytedifferentiation conditions, expressed in a time-dependent fashionprimitive and mature hepatocyte markers, shown by immunofluorescencemicroscopy of double and triple labeled cells. The protein expressionprofile was hepatocyte specific and not spurious, as non-hepatocytemarkers were not co-expressed with hepatocyte antigens. Results fromimmunohistochemistry were confirmed by Western blot. In addition, RT-PCRcorroborated upregulation of the transcription factors HNF3β and GATA4known to be important in endoderm specification and transcriptionfactors required for subsequent hepatocyte differentiation, such asHNF3β, and cytoplasmic proteins such as CK19, CK18, αFP and albumin.

Although it was shown that FGF4 alone or both FGF4 and HGF induced MASCinto cells with morphological and phenotypic characteristics ofhepatocytes, this alone does not prove that cells have differentiatedinto hepatocytes unless one can demonstrate acquisition of functionalcharacteristics of hepatocytes. Therefore, several functional tests weredone in combination to identify functional hepatocytes. mMASC, rMASC orhMASC produced urea and albumin, contained phenobarbital induciblecytochrome P450 activity, could take up Dil-acil-LDL, and containedglycogen granules. Although urea production is characteristic ofhepatocyte activity, kidney tubular epithelium also produces urea(Hedlund E. et al., 2001). In contrast, albumin production is a specifictest for the presence and metabolic activity of hepatocytes (Hedlund E.et al., 2001). Cytochrome P450, although found in hepatocytes, is alsopresent in many other cell types (Jarukamjorn K. et al., 1999). However;Cyp2b1 activity in rat (Tzanakakis E. S. et al., 2001), Cyp2b9 andCyp2b13 in mouse (Li-Masters T. et al., 2001; Zelko L Et al. 2000), andCYP2B6 in human is considered relatively hepatocyte specific. Presenceof these forms of P450 was shown by RT-PCR. The specificity forhepatocytes is enhanced further when P450 activity is inducible byphenobarbital (Rader D J. et al., 2000), as shown. Although LDL uptakeis seen in hepatocytes (Oh S. H. et al., 2000), other cells such asendothelium have a similar capability (Avital I. et al., 2001). Finally,only hepatocytes can generate and store glycogen. When taken together,these functional tests demonstrate that MASC from mouse, rat or humanstreated in vitro with FGF4 and HGF not only express hepatocyte markersbut also have functional characteristics consistent with hepatocytemetabolic activities.

Several studies have shown that BM derived cells may differentiate intohepatocyte-like cells in vivo and in vitro (Petersen B. E. et al., 1999;Theise N. D. et al., 2000; Krause D. S. et al., 2001; Pittenger M. F. etal., 1999; Wang S. et al., 2001; Lagasse E. et al., 2000). However, moststudies have not addressed the phenotype of the BM cell thatdifferentiates into cells with hepatocyte phenotype. It is unknownwhether the cells staining positive for hepatocyte markers hadfunctional characteristics of hepatocytes, and whether cells thatdifferentiate into hepatocytes can also differentiate into mesodermalcells, such as hematopoietic cells. Lagasse et al. demonstrated thatcKit⁺Thy₁ ^(low)Sca1⁺Lin⁺⁻ cells present in murine BM differentiate intocells with not only hepatocyte phenotype but also hepatocyte function(Lagasse E. et al., 2000). Even though such results could be seen whenas few as 50 cells were transplanted, this study did not prove that thesame cell that differentiates into hematopoietic cells alsodifferentiates into hepatocytes. Krause et al showed that a single cellcan repopulate the hematopoietic system and give rise to rarehepatocytes. However, no functional assessment of the hepatocytes wasdone (Krause D. S. et al., 2001). Avital et al recently published thatβ₂m⁻, Thy-1⁺ cells in mouse BM express albumin, HNF4, C/EBPα, andCytochrome P450 3A2 mRNA and protein (Wilmut I., et al., 1997), aphenotype of hepatocyte progenitors usually found in the liver. Thus,presence of such hepatocyte progenitor cells in BM could explain the invivo differentiation of bone marrow into hepatocytes noted in recentstudies (Krause D. S. et al., 2001; Lagasse E. et al., 2000).

To address the question whether cells giving rise to functionalhepatocyte-like cells also give rise to other cell types, retroviralmarking was used (Reyes M. et al., 2001; Jiang Y., 2002). It has beenrecently shown that cells expressing albumin, CK18 and HNF1α can begenerated from the same mMASC and rMASC that differentiate into cellswith endothelial and neuroectodermal phenotype (Jiang Y., 2002). It isconfirmed that similar results are seen for hMASC. Extending recentlypublished studies demonstrating derivation of cells with mesodermal andneuroectodermal phenotype and function from single hMASC (Reyes M.,2002), it is shown here that the same single hMASC also differentiatesinto cells with hepatocyte morphology and phenotype. Thus, it isdemonstrated for the first time that MASC that do not express hepatocytemarkers and have no functional hepatocyte activity exist in BM, whichdepending on the culture conditions, acquire a hepatocyte phenotype andfunctional characteristics of hepatocytes, or phenotypic and functionalcharacteristics of mesodermal and neuroectodermal cells.

Example 12 Transplantation of LacZ Transgenic MASC to Treat HemophiliacMice

MASC were derived from ROSA26 mice containing the β-gal/NEO transgene(10⁶ cells/mouse) and were I.V. injected into hemophiliac mice (N=5)without prior irradiation. The animals were sacrificed at 1 (N=2) and 2months (N=3) post-MASC transplantation. Bone marrow cytospins and frozensections of liver, spleen, skeletal muscle, heart, lung and intestinewere stained for presence of β-gal antigen using a FITC-conjugatedanti-β-gal antibody and pan-cytokeratin or CD45. Tissues were alsoanalyzed by Q-PCR for the β-gal gene as described in Example 6.

Preliminary analysis indicates that one of the three animals (M3)analyzed at 2 months post-injection had 0.1% of pulmonary epithelialcells derived from the donor cells by immunohistochemistry and Q-PCR.immunohistochemistry also showed that animal M5 had <1% engraftment ofCD45⁺ donor cells in the spleen, marrow and intestine. Tissues of theanimal M4 had some donor derived cells on immunohistochemistry; PCR dataon this animal is pending.

All publications, patents and patent applications are incorporatedherein by reference as though individually incorporated by reference.While in the foregoing specification this invention has been describedin relation to certain preferred embodiments thereof, and many detailshave been set forth for purposes of illustration, it will be apparent tothose skilled in the art that the invention is susceptible to additionalembodiments and that certain of the details described herein may bevaried considerably without departing from the basic principles of theinvention.

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What is claimed is:
 1. A method for identifying an agent that affects adesired cellular response, the method comprising: a) contacting anisolated culture-expanded cell population with a desired agent, whereinthe cells of the population are not embryonic stem cells, embryonic germcells, or germ cells, express oct4 and/or telomerase and are nottumorigenic, and have undergone at least 10-40 cell doublings in cultureprior to being contacted with the agent; b) determining whether saidagent affects said cellular response in said cells of the population. 2.A method according to claim 1, wherein the cells of the populationexpress telomerase.
 3. A method according to claim 1, wherein the cellsof the population express oct4.
 4. A method according to claim 2,wherein the cells of the population express oct4.
 5. A method accordingto any one of claims 1-3, wherein the cells of the population candifferentiate into cell types of at least two of the endodermal,ectodermal, and mesodermal embryonic lineages.
 6. A method according toclaim 5, wherein the cells of the population can differentiate into atleast one cell type of each of the endodermal, ectodermal and mesodermalembryonic lineages.
 7. A method according to any one of claims 1-3,wherein the agent is a biological or pharmacological agent.
 8. A methodaccording to claim 7, wherein said biological agent is a cytokine,chemokine, or growth factor.
 9. A method according to any one of claims1-3, wherein said cellular response is gene expression.
 10. The methodof claim 1, wherein the cells that are non-embryonic stem cells,embryonic germ cells, or germ cells have undergone about 30 celldoublings in culture prior to step (a).
 11. The method of claim 1,wherein the cells that are not embryonic stem cells, embryonic germcells, or germ cells, have undergone about 40 cell doublings in cultureprior to step (a).
 12. A method for identifying an agent that affects adesired cellular response, the method comprising: a) contacting anisolated culture-expanded cell population with a desired agent, whereinthe cells of the population are not embryonic stem cells, embryonic germcells, or germ cells, are derived from human bone marrow, express oct4and/or telomerase, and have a normal karyotype, and have undergone atleast 10-40 cell doublings in culture prior to being contacted with theagent; and b) determining whether said agent affects said cellularresponse in said cells of the population.
 13. A method according toclaim 12, wherein the cells of the population express telomerase.
 14. Amethod according to claim 12, wherein the cells of the populationexpress oct4.
 15. A method according to claim 13, wherein the cells ofthe population express oct4.
 16. A method according to any one of claims12-15, wherein the cells of the population can differentiate into celltypes of at least two of the endodermal, ectodermal and mesodermalembryonic lineages.
 17. A method according to claim 16, wherein thecells of the population can differentiate into at least one cell type ofeach of the endodermal, ectodermal and mesodermal embryonic lineages.18. The method of claim 12, wherein the cells that are not non-embryonicstem cells, embryonic germ cells, or germ cells have undergone about 30cell doublings in culture prior to step (a).
 19. The method of claim 12,wherein the cells that are not embryonic stem cells, embryonic germcells, or germ cells, have undergone about 40 cell doublings in cultureprior to step (a).
 20. The method according to claim 1, where the cellsof the population are derived from human bone marrow.